Progenitor cell preservation factors and methods for and products of their use

ABSTRACT

Disclosed is the FRIL family of progenitor cell preservation factors and nucleic acids encoding the same. FRIL family members preserve progenitor cells both in vivo and ex vivo. FRIL family members find use as therapeutics for alleviating and/or reducing the hematopoietic progenitor cell-depleting activity of many cancer therapeutics. FRIL family members are also useful for isolating rare, primitive progenitor cells.

This application is a continuation in part of U.S. Ser. No. 08/881,189,filed Jun. 24, 1997, the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the preservation of progenitor cells. Morespecifically, the invention relates to the in vivo or ex vivopreservation of progenitor cells, such as hematopoietic progenitorcells.

2. Summary of the Related Art

The wide variety of functionally and phenotypically different types ofcells in a multi-cellular eukaryotic organism results in part from theproliferation and differentiation of rare and mostly quiescentpopulations of progenitor cells. For example, hematopoiesis involves theprocess of producing a balanced supply of different blood cells fromsuch progenitor cells found in the adult bone marrow. The development ofother cell types also depends upon production of the differentiatedcells from such progenitor cells.

Progenitor cells are activated by signals, such as cell-cell contact orsoluble regulators, to generate daughter cells that are identical to theparent (i.e., self-renewal of the parent) and/or to generate daughtercells that are more differentiated than the parent, thus beginning anirreversible process that ends with the production of differentiated,functional cells. In the process of hematopoiesis, differentiation iscoupled to proliferation as a progenitor cell gives rise to moredifferentiated daughter cells that progressively become committed toproducing only one blood cell type. The enormous activation ofhematopoietic progenitor cells needed to meet the body's dailyrequirement for hundreds of billions of new mature blood cells isdirected by potent soluble regulators (e.g., colony stimulating factorsand cytokines) acting upon the hematopoietic progenitor cellsthemselves, and their more differentiated daughter cells.

Although progenitor cells eventually produce so many of the mature cellsof the body, they occur only rarely. Moreover, typically the moreprimitive (i.e., undifferentiated) the progenitor cell, the more rarethe progenitor cell. For example, the currently believed most primitiveof the hematopoietic progenitor cells, which are called hematopoieticstem cells, occur at a frequency of only from about 1 in 10,000 to about1 in 100,000 of the cells in the bone marrow. Hematopoietic stem cellshave the capacity to generate more than 10¹³ mature blood cells of alllineages, including other progenitor cells which, although moredifferentiated than hematopoietic stem cells, are themselves capable ofgiving rise to several different types of mature blood cells.

Hematopoietic stem cells are responsible for sustaining blood cellproduction over the life of an animal. The small population ofhematopoietic stem cells is sufficient to produce all the mature bloodcells in a healthy individuals; however, some unhealthy individualssuffer from a lack of a sufficient number of progenitor cells and/ormature blood cells. For example, cancer patients receivingchemotherapeutic or radiotherapy treatments designed to kill the rapidlydividing cancer cells also suffer from the depletion of white bloodcells and platelets, thus exposing these patients to life threateningopportunistic infections and bleeding episodes. Indeed, thishematopoietic progenitor cell-depleting activity is the dose-limitingfactor for most of these chemotherapeutic and radiotherapeutic agents.

Many cancer patients are routinely treated with cytokines, includingG-CSF, GM-CSF, SCF, Erythropoietin, and IL-11, to accelerate restorationof hematopoiesis following chemotherapy (Moore, M. A., Blood 78: 1-19,1991). However, these cytokines lead to the irreversible differentiationof hematopoietic progenitor cells, including hematopoietic stem cells,into more differentiated daughter cells. Thus, better protection ofhematopoietic progenitor cells is needed during chemotherapy.

Workers in the field have attempted to use cytokines in mice to protectprogenitors from the toxicity of chemotherapy (Neta et al., J. Immunol.136: 2483-2485, 1986; Neta et al., J. Immunol. 140: 108-111, 1988; Netaet al., J. Exp. Med. 173: 1177-1182, 1991; de Haan et al., Blood 87:4581-4588, 1996; Lyman and Jacobsen, Blood 91: 1101-1134, 1998; Dalmauet al., Bone Marrow Transplant. 12: 551-563, 1993; Grzegorzewski et al.,J. Exp. Med. 180: 1046-1057, 1994; Grzegorzewski et al., Blood 94:1066a(Abstr.)1999). Marshall et al. (Euro. J. Cancer 34: 1023-1029, 1998) andGilmore et al. (Exp. Hematol. 27: 195-202, 1999) describe the use inclinical trials of a chemokine that allegedly inhibits progenitor cellproliferation, MIP1-α, as a chemoprotectant. Marshall (Marshall, A.,Nat.. Biotechnol. 16: 129, 1999) describes the use of MPIF-1, achemokine that allegedly inhibits progenitor cell proliferation, inclinical trials as a chemoprotectant.

There are several drawbacks to using chemokines, cytokines, and otherimmunoregulators as chemoprotectants during the chemotherapeutic orradiotherapeutic treatment of cancer patient. These drawbacks includethe cost of production and toxicity to the patient.

Therefore, there is a need for improved reagents that are non-toxic andinexpensive to produce for use in preserving progenitor cells.

BRIEF SUMMARY OF THE INVENTION

The invention provides a family of factors that preserve progenitorcells. Members of this family, the FRIL family, are non-toxic,inexpensively produced reagents that preserve progenitor cells. Theinvention provides compositions comprising at least one member of theFRIL family, as well as methods for using members of the FRIL family topreserve progenitor cells both in vivo and ex vivo.

Accordingly, in a first aspect, the invention provides an essentiallypure composition of one or more members of the FRIL family of progenitorcell preservation factors.

In certain embodiments of the first aspect of the invention, the FRILfamily member is from a legume. In some embodiments, the legume isDolichos lab lab. In some embodiments, the legume is Phaseolus vulgaris.In some embodiments, the legume is Sphenostylis stenocarpa.

In certain embodiments of the first aspect of the invention, the FRILfamily member is a mutant derived from a second member of the FRILfamily, wherein the mutant is selected from the group consisting of asubstitution mutant, a deletion mutant, an addition mutant, or acombination thereof.

In certain embodiments of the first aspect of the invention, the FRILfamily member is a fusion protein comprising a first portion and asecond portion, wherein the first portion is derived from a secondmember of the FRIL family.

In a second aspect, the invention provides a recombinant nucleic acidmolecule encoding a composition of a member of the FRIL family ofprogenitor cell preservation factors.

In a third aspect, the invention provides a pharmaceutical formulationcomprising an essentially pure composition of one or more members of theFRIL family of progenitor cell preservation factors and apharmaceutically acceptable carrier.

In certain embodiments of the third aspect of the invention,administration of a therapeutically effective amount of the formulationto a patient suffering from a condition whereby the patient'shematopoietic progenitor cells are depleted alleviates and/or reducesthe condition in the patient.

In certain embodiments of the third aspect-of the invention,administration of a therapeutically effective amount of the formulationto a patient prior to treatment of the patient with a therapeutictreatment having a hematopoietic progenitor cell-depleting activityalleviates and/or reduces the hematopoietic progenitor cell-depletingactivity of the therapeutic treatment in the patient. In certainembodiments, the patient is a human or is a domesticated mammal. In someembodiments, the patient has cancer. In some embodiments, thetherapeutic treatment is a radiotherapeutic or a chemotherapeutictreatment, including, without limitation, cytarabine (Ara-C),doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combination of aradiotherapeutic and a chemotherapeutic.

In a fourth aspect, the invention provides a method for alleviating orreducing the hematopoietic progenitor cell-depleting activity of atherapeutic treatment in a patient, comprising administering to theanimal a therapeutically effective amount of a composition of a FRILfamily member prior to administration of the therapeutic treatment tothe patient.

In certain embodiments of the fourth aspect, the patient is a human oris a domesticated mammal. In some embodiments, the patient has cancer.In some embodiments, the therapeutic treatment is a radiotherapeutic ora chemotherapeutic treatment, including, without limitation, cytarabine(Ara-C), doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combinationof a radiotherapeutic and a chemotherapeutic.

In a fifth aspect, the invention provides a method for isolating apopulation of progenitor cells, comprising contacting a population ofcells with a plurality of FRIL family member molecules, and separatingthe unbound cells, wherein the cells bound to the FRIL family membermolecules are an isolated population of progenitor cells. Preferably,the isolated population of progenitor cells is from a human.

In certain embodiments of the fifth aspect of the invention, the FRILfamily member molecules are detectably labeled. In certain embodiments,the detectably labeled FRIL family member molecules are labeled with achromophore. In certain embodiments, the unbound cells are separated byusing a flow cytometry cell sorter to sort the population of cellscontacted with the FRIL family member molecules detectably labeled witha chromophore.

In certain embodiments of the fifth aspect, the FRIL family membermolecules is immobilized on a solid support. In some embodiments, thesolid support is a bead, such as a magnetic bead. In some embodiments,the unbound cells are separated by applying a magnet to the populationof cells contacted with the FRIL family member molecules immobilized onthe magnetic bead. In further embodiments, the population of cells boundto the FRIL family member molecules immobilized on a magnetic bead isrinsed with a physiologically acceptable solution while the magnet isapplied.

In certain embodiments of the fifth aspect, the solid support is thebottom of a tissue culture plate. In some embodiments, the unbound cellsare separated by rinsing the population of cells contacted with the FRILfamily member immobilized on the bottom of a tissue culture plate with aphysiologically acceptable solution.

In preferred embodiments of the fifth aspect of the invention, theisolated population of progenitor cells is a population of hematopoieticprogenitor cell. In various embodiments, the population of cells iswhole blood, umbilical cord blood, bone marrow cells, or fetal livercells.

In certain embodiments of the fifth aspect of the invention, thepopulation of cells is a sorted population of cells, wherein a cell ofthe sorted population does not express a cell surface molecule selectedfrom the group consisting of CD11b, CD11c, and CD38. In certainembodiments, the sorted population of cells is sorted by flow cytometryor by magnetic bead selection.

In a sixth aspect, invention provides an isolated population ofprogenitor cells isolated by a method comprising contacting a populationof cells with a plurality of FRIL family member molecules, andseparating the unbound cells, wherein the cells bound to the FRIL familymember molecules are an isolated population of progenitor cell.Preferably, the progenitor cell is from a human.

In certain embodiments of the sixth aspect, the cells of the isolatedpopulation do not express CD34. In certain embodiments, the cells of theisolated population express a receptor tyrosine kinase selected from thegroup consisting of from FLK1, FLT1, FLT3, FLT4, and Kit. In someembodiments, the cells of the isolated population express a cell surfacemolecule selected from the group consisting of CD11b and CD11c. Inpreferred embodiments, the cells of the isolated population expressFLT3.

In various embodiments of the sixth aspect of the invention, the cellsof the isolated population are hemangioblasts, messenchymal stem cells,bone progenitor cells, hepatic progenitor cells, endothelial progenitorcells, hematopoietic progenitor cells, embryonal stem cells, brainprogenitor cells, or dendritic progenitor cells. Preferably, the cellsof the isolated population are hematopoietic progenitor cells.

In certain embodiments of the sixth aspect of the invention, where thecells of the isolated population are hematopoietic progenitor cells,transplantation of the isolated population into an animal lacking apopulation of hematopoietic progenitor cells sufficient to enablesurvival of the animal reconstitutes the animal, wherein thetransplanted animal survives. In certain embodiments, the hematopoieticprogenitor cells are from a human or a mouse and wherein the animal is amouse. In some embodiments, the mouse is a SCID mouse or the mouse issublethally irradiated or the mouse is treated with a sublethal dose ofa chemotherapeutic. In certain embodiments, the hematopoietic progenitorcells are from a human and the animal is a human. In some embodiments,the human is a cancer patient receiving a treatment that depletes thepatient's hematopoietic progenitor cells. In some embodiments, thetreatment is a radiotherapeutic or a chemotherapeutic treatment,induding, without limitation, cytarabine (Ara-C), doxorubicin (Dox), or5-fluorouracil (5-FU), or a combination of a radiotherapeutic and achemotherapeutic.

In a seventh aspect, the invention provides a method for preservingprogenitor cells ex vivo comprising contacting a population of cellscomprising at least one progenitor cell with an effective amount of acomposition of a FRIL family member for an effective period of time,wherein the progenitor cells in the population are rendered quiescent.

In certain embodiments of the seventh aspect, the progenitor cells arefrom a human. In certain embodiments, the population of cells is bonemarrow cells. In some embodiments, the non-progenitor cells in thepopulation of cells differentiate or die.

In certain embodiments of the seventh aspect, the population of cells isremoved from a cancer patient prior to treatment of the cancer patientwith a therapeutic treatment having a hematopoietic progenitorcell-depleting activity. In some embodiments, the therapeutic treatmentis a radiotherapeutic or a chemotherapeutic treatment, including,without limitation, cytarabine (Ara-C), doxorubicin (Dox), or5-fluorouracil (5-FU), or a combination of a radiotherapeutic and achemotherapeutic.

In an eighth aspect, the invention provides a method for preservingprogenitor cells in vivo, comprising administering to a patient aneffective amount of a composition of a FRIL family member for aneffective period of time, wherein the progenitor cells in the patientare rendered quiescent. Preferably, the patient is a human or adomesticated animal.

In certain embodiments of the eighth aspect of the invention, thepatient is a cancer patient. In some embodiments, the effective amountof the composition of a FRIL family member is administered prior to thetreatment of the patient with a therapeutic treatment having ahematopoietic progenitor cell-depleting activity. In some embodiments,the therapeutic treatment is a radiotherapeutic or a chemotherapeutictreatment, including, without limitation, cytarabine (Ara-C),doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combination of aradiotherapeutic and a chemotherapeutic.

In a ninth aspect, the invention provides a method for identifying aprogenitor cell, comprising contacting a candidate cell with a FRILfamily member molecule, wherein binding of the candidate cell to theFRIL family member molecule identifies the candidate cell as aprogenitor cell.

In certain embodiments of the ninth aspect, the candidate cell is in apopulation of cells. In certain embodiments, the candidate cell is froma human.

In a tenth aspect, the invention provides a progenitor cell identifiedby a method comprising contacting a candidate cell with a FRIL familymember molecule, wherein binding of the candidate cell to the FRILfamily member identifies the candidate cell as a progenitor cell.

In an eleventh aspect, the invention provides a method for identifying acomposition of a member of the FRIL family of progenitor cellpreservation factors, comprising contacting a candidate compound with aglycosylated extracellular domain of an FLT3 receptor, wherein theglycosylation pattern of the extracellular domain of the FLT3 receptoris the same as the glycosylation pattern of an extracellular domain of anormally glycosylated FLT3 receptor, wherein a candidate compound thatbinds the glycosylated extracellular domain of the FLT3 receptor isidentified as a composition of a FRIL family member.

In certain embodiments of the eleventh aspect, the candidate compound isa lectin. In certain embodiments, the lectin is synthetic. In certainembodiments, the lectin is from a legume.

In a twelfth aspect, the invention provides an essentially purecomposition of a FRIL family member identified by the method comprisingcontacting a candidate compound with a glycosylated extracellular domainof an FLT3 receptor, wherein the glycosylation pattern of theextracellular domain of the FLT3 receptor is the same as theglycosylation pattern of an extracellular domain of a normallyglycosylated FLT3 receptor, wherein a candidate compound that binds theglycosylated extracellular domain of the FLT3 receptor is identified asa member of the FRIL family.

According to the invention, compositions of a FRIL family member may beused as therapeutic agents to preserve progenitor cells in patients,such as cancer patients receiving chemotherapy, who have suffer from acondition that diminishes their progenitor cells. For example,compositions of a FRIL family member may be administered with apharmaceutically-acceptable carrier (e.g., physiological sterile salinesolution) via any route of administration to a cancer patient receivingchemotherapy in an attempt to reduce the progenitor cell-depletingeffects of the chemotherapeutic so that the patient can receive a higherdose of the chemotherapeutic and, preferably, recover from cancer.Pharmaceutically-acceptable carriers and their formulations arewell-known and generally described in, for example, Remington'sPharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of a cloning vector pCR2.1-DLA manufactured by ligatinga cDNA according to the invention in the EcoRI site of the cloningvector pCR2.1.

FIG. 2 shows a direct amino acid sequence comparison of the mannoselectin described by Gowda et al. (J Biol Chem 269:18789-18793, 1994) andthe derived amino acid sequence of Dl-FRIL, a representative,non-limiting FRIL family member of the invention, encoded by arepresentative, non-limiting nucleic acid of the invention.

FIG. 3 is a map of a cloning vector pCR2.1-DLA(D) manufactured byligating a mutated cDNA in the EcoRI site of the cloning vector pCR2.1.

FIG. 4 is a map of a cloning vector pBS-SpDLA manufactured by ligating arecombinant fragment in the EcoRI site of the cloning vector pBluescriptSK+.

FIG. 5 is a map of a cloning vector pCR2.1-SpM1(D) manufactured byligating a mutated recombinant clone in the EcoRI site of the cloningvector pCR2.1.

FIG. 6 is a map of a recombinant expression vector pBIN-VicPromanufactured by subcloning the vicilin promoter obtained from the pCW66vector in the EcoRI/ClaI site of the plant binary vector pBIN19 forAgrobacterium-mediated transformation.

FIG. 7 is a map of a recombinant expression vector pBINVicPro-SpDLAmanufactured by ligating a recombinant fragment in the EcoRI/SacI siteof the pBINVicPro vector.

FIG. 8 is a map of a recombinant expression vector pBINVicPro-SpDLA(D)manufactured by ligating a mutated recombinant clone in the EcoRI siteof the pBINVicPro vector.

FIG. 9 is a map of a recombinant expression vector pGEX4T-1-DLAmanufactured by ligating a wild-type cDNA clone in the EcoRI/SalI siteof the E. coli expression vector pGEX4T-1.

FIG. 10 is a map of a recombinant expression vector pGEX4T-1-DLA(D)manufactured by ligating a mutant cDNA clone in the EcoRI/XhoI site ofthe E. coli expression vector pGEX4T-1.

FIG. 11 is a representation of an electrophoretogram of a Southern blotof total protein extracts of E. coli cells transformed with therecombinant expression vectors pGEX4T-1-DLA and pGEX4T-1-DLA(D).

FIG. 12 is a representation of an electrophoretogram of a Western blotof purified GST-fusion proteins with and without cleavage by thrombin.

FIG. 13A is a representation of a graph showing that a crude extract ofan E. coli culture containing expressed Dl-FRIL, a representative,non-limiting FRIL family member of the invention, specificallystimulates hFLT3 3T3 cells; FIG. 13B is a graph showing that the sameextract does not stimulate untransfected 3T3 cells.

FIG. 14A is a representation of a histogram showing that purifiedDl-FRIL, a representative, non-limiting FRIL family member of theinvention, preserves cord blood mononudear cells in a dose-responsivemanner.

FIG. 14B is a representation of a histogram showing that purifiedDl-FRIL, a representative, non-limiting FRIL family member of theinvention, preserves hematopoietic progenitors in a dose-responsivemanner.

FIG. 15A is a representation of a photograph of colonies derived fromhuman cord blood mononuclear cells cultured in 40 ng/mL Dl-FRIL, arepresentative, non-limiting FRIL family member of the invention, for 3weeks, and then replated in methylcellulose colony assay medium.

FIG. 15B is a representation of a photograph of colonies derived fromhuman cord blood mononuclear cells cultured in 40 ng/mL Dl-FRIL, arepresentative, non-limiting FRIL family member of the invention, for 4weeks, and then replated into methylcellulose colony assay medium.

FIG. 16 is a schematic diagram showing the serial replating ofprogenitor cells cultured in Dl-FRIL, a representative, non-limitingFRIL family member of the invention, or Dl-FRIL+recFL (i.e., recombinantFLT3-Ligand). The human cord mononuclear cells were first cultured insupensiion in 40 ng/mL Dl-FRIL of 40 ng/mL D1-FRIL+40 ng/mL recFL (solidblack box). The cells were then harvested and assessed for progenitoractivity by being replated into methylcellulose colony assay medium for6 weeks (middle striped box). Then the cells were harvested from thecolony assay and again replated into methylcellulose colony assay mediumfor an additional 4 weeks (far right striped box). Progenitorfrequencies were determined for cells after 3 weeks of suspensionculture in Dl-FRIL or Dl-FRIL+recFL, and after an additional 6 weeks ofmethylcellulose culture (absent Dl-FRIL and/or recFL).

FIG. 17 is a representation of a photograph of colonies derived fromhuman cord blood mononuclear cells initially cultured in 40 ng/mLDl-FRIL, a representative, non-limiting FRIL family member of theinvention, for 3 weeks, then replated into methylcellulose colony assaymedium for 6 weeks, and then replated again into methylcellulose colonyassay medium for an additional 4 weeks.

FIG. 18 is a representation of a bar graph showing that arepresentative, non-limiting FRIL family member of the invention,Dl-FRIL, encoded by a representative, non-limiting nucleic acid of theinvention is sufficient to preserve progenitor cells ex vivo, whereas acytokine cocktail fails to preserve such cells.

FIGS. 19A and 19B are representations of line graphs showing thebiological specificity of receptor-transfected 3T3 cells. FIG. 19A showsthat rhM-CSF specifically stimulated Fms 3T3 (solid circles) but noteither mFlt3/Fms 3T3 (open circles) or parent 3T3 cells (solid squares)in biological screening assay in a dose-dependent manner. FIG. 19B showsthat PHA-LCM (reciprocal dilution) stimulated mFlt3/Fms 3T3 (solidcircles) and Stk 3T3 (open circles) but not parent untransfected 3T3cells (solid squares).

FIGS. 20A-20D are representations of the detected biological activitiesof PHA-LCM fractionated by anion exchange chromatography. FIG. 20A showsthe number of viable cells cord blood cells observed microscopically.FIG. 20B shows the stimulation of mFlt3/Fms 3T3 cells by anion exchangecolumn fractions. FIG. 20C shows the stimulation of Stk 3T3 cells byanion exchange column fractions. FIG. 20D shows the stimulation of Fms3T3 cells by anion exchange column fractions.

FIGS. 21A and 21B are representations of line graphs showing theco-factors required in the Flt3 3T3 assay during purification ofPv-FRIL, a representative, non-limiting FRIL family member of theinvention. FIG. 21A shows that the plateau stimulation of Flt3 3T3 cellsdecreased during purification from crude, 10-fold concentrated PHA-LCM(solid circles) to partially purified (open circles), and highlypurified (solid squares). Medium control is shown is open squares. FIG.21B shows that the decreased plateau stimulation of Flt3 3T3 cells(solid circles) was restored by addition of sub-optimal concentrations(1:200) of crude PHA-LCM (solid squares). Corresponding medium controlsare shown in open symbols.

FIGS. 22A-22D are representations of a series of flow cytometryhistograms showing the re-analysis of CD34 expression of cord bloodCD34⁺ cells after two weeks in suspension culture with pooled AQSaffinity column fractions. CD34 expression was re-analyzed by flowcytometry on pooled AQS affinity column fractions 1-5 (FIG. 22A),fractions 6-10 (FIG. 22B), fractions 11-15 (FIG. 22C), and fractions16-20 (FIG. 22D). Fluorescence intensity on the abcissa and frequency ofevents on the ordinate in FIGS. 22A-22D.

FIG. 23 is a representation of a line graph showing the IL1-dependentresponse of mFlt3/Fms 3T3 cells to FRIL isolated from commercial redkidney bean extract. mFlt3/Fms 3T3 cells respond to FRIL in the presenceof rhIL1 (solid circles) but not the absence of IL1 (solid squares).Corresponding medium only controls are shown with open symbols.

FIG. 24A is a map of a cloning vector pCR2.1-Pv-FRIL manufactured byligating a cDNA according to the invention in the EcoRI site of thecloning vector pCR2.1.

FIG. 24B shows a direct amino acid sequence comparison of Pv-FRIL, arepresentative, non-limiting FRIL family member of the invention, withDl-FRIL, another representative, non-limiting FRIL family member of theinvention, and the PHA lectin, PHA-E.

FIG. 25 is a map of a cloning vector pCR2.1-SpPv-FRIL manufactured byligating a cDNA according to the invention in the Xhol site of thecloning vector pCR2.1.

FIG. 26 is a map of a cloning vector pM-SpPv-FRIL manufactured byligating a cDNA according to the invention in the Bg1II/Xhol sites ofthe cloning vector SpPv-FRIL.

FIGS. 27A-27B are representations of line graphs showing the total cellnumbers and progenitor levels in the presence of Dl-FRIL,arepresentative, non-limiting FRIL family member of the invention, orcytokines. Enriched CB CD34⁺ cells were cultured for 3, 6, 10, or 13days in the presence of Dl-FRIL (solid symbols) or cytokines (opensymbols). Colonies were scored on day 14 and progenitor levels werecalculated based on total cell numbers. Values shown represent themean±SEM of data from up to 10 experiments. FIG. 27A shows the totalcell numbers over time. FIG. 27B shows the progenitor levels in culturesover time.

FIGS. 28A-28D are representations of line and bar graphs showing thetotal cell numbers and progenitor levels first in the presence ofDl-FRIL, a representative, non-limiting FRIL family member of theinvention, and second in presence of cytokines. FIG. 28A shows the totalnumbers of cells cultured with Dl-FRIL for entire 10 days (solidsymbols) or for 6 days followed by 4 days of cytokine stimulation (opensymbols). FIG. 28B shows the progenitor levels in cells cultured withDl-FRIL for entire 10 days (solid symbols) or for 6 days followed by 4days of cytokine stimulation (open symbols). FIG. 28C shows the totalnumbers of cells cultured with Dl-FRIL for 13 days (solid symbols) orfor 10 days followed by 3 days of cytokine stimulation (open symbols).FIG. 28D shows the progenitor levels in cells cultured with Dl-FRIL for13 days (solid symbols) or for 10 days followed by 3 days of cytokinestimulation (open symbols).

FIGS. 29A-29D are representations of representative Southern blotanalyses showing the quantitative analysis of SRC after ex vivo cultureswith Dl-FRIL,a representative, non-limiting FRIL family member of theinvention, or cytokines and after transplantation into mice. FIG. 29A isa representation of a representative Southern blot showing human DNA inthe marrow of mice transplanted with cells that were cultured withDl-FRIL for 6 days (lane 1), Dl-FRIL for 10 days (lane 2), or withDl-FRIL for 6 days followed by 4 days with cytokine stimulation (lane3). FIG. 29B is a representation of a representative Southern blotshowing human DNA in the marrow of mice transplanted with cells culturedwith Dl-FRIL for 10 days (lanes 1-2), or with Dl-FRIL for 6 daysfollowed by 4 days with cytokine stimulation (lanes 34). FIG. 29C is arepresentation of a representative Southern blot showing human DNA inthe marrow of mice transplanted with the original cells prior to seeding(lane 1), or with cells cultured with Dl-FRIL for 13 days (lane 2). FIG.29D is a representation of a representative Southern blot showing humanDNA in the marrow of mice transplanted with cells cultured with FRIL for10 days (lane 1), Dl-FRIL for 6 days followed by 4 days of cytokinestimulation (lane 2), Dl-FRIL for 13 days (lane 3), or with Dl-FRIL for10 days followed by 3 days of cytokine stimulation (lane 4).

FIG. 29E is a representation of a Southern blot analysis showing thedetection of a 0%; 0.1%, 1%, and 10% human DNA per murine DNA.

FIG. 30 is a representation of a survival chart summarizing the levelsof human cell engraftment in the marrow of mice transplanted with CD34⁺cells cultured in either Dl-FRIL, a representative, non-limiting FRILfamily member of the invention, for ten days (left panel), or in thepresence of Dl-FRIL for 6 days followed by culture in the presence ofcytokines for four days.

FIG. 31 is a representation of a representative Southern blottinganalysis showing the levels of levels of human cell engraftment in theBM of NOD/SCID B2M^(null) transplanted with CD34⁺CD38^(−/low) cellscultured in the presence of Dl-FRIL, a representative, non-limiting FRILfamily member of the invention. Sorted cells (2×10⁵ initialcells/treatment) were cultured in the presence of Dl-FRIL for 6 daysfollowed by additional 4 days exposure to cytokines, or with Dl-FRILalone for 10 days. After 10 days, 3.6×10⁵ cells harvested from cytokineculture were divided and transplanted into 3 mice (lanes 1-3), while3.5×10⁴ cells harvested from Dl-FRIL alone culture were transplanted toone mouse (lane 4). DNA was harvested from the bone marrow oftransplanted mice and subjected to Southern blotting analysis withradiolabeled human chromosome 17-specific α-satellite probe (p17H8). Arepresentative experiment out of 4 is shown.

FIG. 32 is a representation of a bar graph showing the average foldincrease of engraftment levels obtained with CD34⁺CD38^(−/low) cellsthat were cultured with Dl-FRIL, a representative, non-limiting FRILfamily member of the invention, for 6 days and with cytokines for 4 days(right bar), as compared to 10 days with Dl-FRIL alone (left bar). Datarepresent mean±SE from 4 experiments.

FIGS. 33A-33F are representations of a bar graph (FIG. 33A) andrepresentative flow cytometry histograms (FIG. 33B-33F) showing themultilineage differentiation of SRC cultured with Dl-FRIL, arepresentative, non-limiting FRIL family member of the invention, in theBM of transplanted mice. FIG. 33A shows the number of colonies per 2×10⁵cells, where the cells treated as indicated prior to transplantationinto mice were recovered from the murine BM, and seeded into semisolidmedia selective for human colonies. Progenitor levels were calculatedbased on the total human cell numbers (2×10⁵ cells) in the marrow oftransplanted mice. Values shown represent the mean±SEM of data from 3experiments, 9 mice/treatment. FIG. 33B shows a representative flowcytometry analysis showing nonspecific labeling where mouse IgG was usedas isotype control. FIGS. 33C and 33D show representative flow cytometryanalyses of BM cells from mice that were transplanted with CD34⁺ cells(FIG. 33C) or CD34⁺CD38^(−/low) cells (FIG. 33D) precultured withDl-FRIL for 10 days, where the harvested BM cells were stained with antihuman CD45 and anti-human CD19. FIGS. 33E and 33F show representativeflow cytometry analyses of BM cells from mice that were engrafted byCD34⁺ cells (FIG. 33E) or CD34⁺CD38^(−/low) cells (FIG. 33F) preculturedwith Dl-FRIL for 10 days, where the harvested BM cells were subsequentlycultured with SCF+IL-15 for 10 days, and then stained with humanspecific monoclonal anti-CD45 and anti-CD56.

FIGS. 34A-34B are representations of bar graphs showing the growtheffect of Dl-FRIL, a representative, non-limiting FRIL family member ofthe invention, on CD34⁺ cells and progenitors compared to Flt3 ligandand cytokine combinations. (+) indicates co-culture of factors for theentire 10 days while (→) indicates substitution of the first factorafter 6 days with cytokines, as indicated under the x axis. FIG. 34Ashows the total cell numbers. FIG. 34B shows the percentage of CFU-GEMMout of total colonies. Values shown are per 2×10⁵ seeded cells, andrepresent the mean±SEM of data from 5 experiments.

FIGS. 35A-35G are representatives of flow cytometry histograms showingthe cell cycle analyses of CD34⁺ cells cultured with Dl-FRIL, arepresentative, non-limiting FRIL family member of the invention, orvarious cytokines, or with combinations thereof. DNA content wasdetermined by flow cytometry with propidium iodide staining. EnrichedCD34⁺ cells were cultured for 3 days with no treatment (FIG. 35A),Dl-FRIL (FIG. 35B), Flt3-L (FIG. 35C), Dl-FRIL+Flt3-L (FIG. 35D),SCF+G−CSF+IL-3+IL-6 (SG36) (FIG. 35E), SG36+Dl-FRIL (FIG. 35F), andSG36+Flt3-L (FIG. 35G).

FIGS. 36A-36C are representations of line graphs showing the doseresponse of CB mnc chemotherapeutic agents in the presence and absenceof Dl-FRIL, a representative, non-limiting FRIL family member of theinvention. Chemotherapy agents were assayed over a 5-log dose range onCB MNC (2×10⁵ cells/0.1 mL) in AIMV (Life Technologies) containingAgar-SCM (StemCell Technologies). Viable cells were determined after 5days of culture by XTT. Solid squares indicate chemotherapy drug with noDl-FRIL; solid triangles indicate cultures containing Dl-FRIL at 10ng/ml in all wells; and open circles indicate Dl-FRIL in all wells at100 ng/ml. FIG. 36A shows the dose response to Ara-C; FIG. 36B shows thedose response to doxorubicin, and FIG. 36C shows the dose response to5-FU.

FIG. 37 is a representation of a photograph of purified Dl-FRIL, arepresentative, non-limiting FRIL family member of the invention,resolved by SDS-PAGE analysis. Five discrete bands appeared whichcorresponded to the α and β subunits, each of which was subjected toamino-terminal sequencing. The amino acid sequences of the five bandsare as indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates the preservation of progenitor cells by members ofthe FRIL family of progenitor cell preservation factors. Morespecifically, the invention relates to the in vivo or ex vivopreservation of progenitor cells, such as hematopoietic progenitorcells, using members of the FRIL family of progenitor cell preservationfactors.

The invention provides a family of factors that preserve progenitorcells. Members of this family, the FRIL family, are non-toxic,inexpensively produced reagents that preserve progenitor cells. Theinvention provides compositions comprising at least one member of theFRIL family, as well as methods for using members of the FRIL family topreserve progenitor cells both in vivo and ex vivo.

All of the patents and publications cited herein reflect the knowledgein the art and are hereby incorporated by reference in entirety to thesame extent as if each were specifically stated to be incorporated byreference. Any inconsistency between these patents and publications andthe present disclosure shall be resolved in favor of the presentdisclosure.

In a first aspect, the invention provides an essentially purecomposition of a member of the FRIL family of progenitor cellpreservation factors. The term, “FRIL family of progenitor cellpreservation factors” is used to mean a family of lectins, wherein eachFRIL family member molecule binds to a normally glycosylated FLT3receptor, wherein each FRIL family member molecule preserves progenitorcells, and wherein one FRIL family member molecule that is isolated froma hyacinth bean (i.e., Dolichos lab lab) has an amino acid sequencewhich comprises the following eight amino acid sequence: TNNVLQXT (SEQID NO: 24). By “FRIL family member” or FRIL family member molecule” ismeant one or more molecules of the FRIL family of progenitor cellpreservation factors.

In accordance with the first aspect of the invention, a composition of aFRIL family member, which includes a mutant of another FRIL familymember molecule or a fusion protein comprising a portion derived from aFRIL family member molecule or mutant thereof, wherein each FRIL familymember molecule binds to a normally glycosylated FLT3 receptor has atleast about 45% amino acid sequence identity with the amino acidsequence of another member of the FRIL family, preferably at least about50% identity, even more preferably at least about 55% identity, stillmore preferably at least about 60% identity, and still more preferablyat least about 65% identity with the sequence of the second protein. Inthe case of proteins having high sequence identity, the amino acidsequence of the first protein shares at least about 75% sequenceidentity, preferably at least about 85% identity, and more preferably atleast about 95% identity, with the amino acid sequence of another memberof the FRIL family.

Both amino acid sequence identity and nucleic acid sequence identitybetween two proteins or two nucleic add molecules can be measuredaccording to standard methods. For example, in order to compare a firstamino acid sequence to a second amino acid sequence or a first nucleicacid sequence to a second nucleic acid sequence for the purpose ofdetermining percentage identity between the two sequences, the sequencesare aligned so as to maximize the number of identical amino acid ornucleic acid residues. The sequences of proteins sharing at least 50%amino add sequence identity or the sequences of nucleic acids sharing atleast 45% nucleic acid sequence identity can usually be aligned byvisual inspection. If visual inspection is insufficient, the proteins ornucleic acids may be aligned in accordance with the FASTA method inaccordance with Pearson and Lipman (Proc. Natl. Acad. Sci. USA85:2444-2448, 1988), or, preferably, any of the methods described byGeorge, D. G. et al., in Macromolecular Sequencing and Synthesis,Selected Methods and Applications, pages 127-149, Alan R. Liss, Inc.(1988), such as formula 4 at page 137 using a match score of 1, amismatch score of 0, and a gap penalty of −1. From this method,percentage of sequence identity between the first and second amino acidsequences or between the first and second nucleic acid can bedetermined.

Other methods for determining amino acid or nucleic acid sequenceidentity are described in Feng and Doolittle (Journal of MolecularEvolution 25: 351-360, 1987) and Higgins and Sharp (CABIOS 5:151-153,1989).

Another method for determining amino acid or nucleic acid sequenceidentity between two proteins or nucleic acids is by using sequenceanalysis software with the default parameters specified therein. Varioussoftware packages exist including Sequence Analysis Software Package ofthe Genetics Computer Group (University of Wisconsin BiotechnologyCenter, Madison, Wis.), and the various BLAST programs of the NationalCenter for Biotechnology (National Library of Medicine, Bethesda, Md.).

Unless otherwise specified, percentage of amino acid sequence identityor percentage of nucleic acid sequence identity is determined using thebasic BLAST program of the National Center for Biotechnology (NationalLibrary of Medicine, Bethesda, Md.), using the default settings definedtherein.

Another test for percentage identity of two nucleic acid sequences iswhether they hybridize under normal hybridization conditions, preferablyunder stringent hybridization conditions. Thus, also included in theinvention are proteins that are encoded by nucleic acid molecules thathybridize under high stringent conditions to a sequence complementary toSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and/or SEQ ID NO: 7. The term“stringent conditions,” as used herein, is equivalent to “high stringentconditions” and “high stringency.” These terms are used interchangeablyin the art.

Stringent conditions are defined in a number of ways. In one definition,stringent conditions are selected to be about 50° C. lower than thethermal melting point (T_(m)) for a specific sequence at a defined ionicstrength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched sequence. Typical stringent conditions are those inwhich the salt concentration is at least about 0.02 M at pH 7 and thetemperature is at least about 60° C. “Stringent conditions,” inreferring to percentage identity (e.g., homology) or substantialsimilarity in the hybridization context, can be combined conditions ofsalt, temperature, organic solvents or other parameters that aretypically known to control hybridization reactions. The combination ofparameters is more important than the measure of any single parameter.If incompletely complementary sequences recognize each other under highstringency conditions, then these sequences hybridize under conditionsof high stringency (see U.S. Pat. No. 5,786,210; Wetmur and Davidson, J.Mol. Biol. 31, 349-370, 1968). Control of hybridization conditions, andthe relationships between hybridization conditions and degree ofhomology are understood by those skilled in the art. See, e.g., Sambrooket al., Molecular Cloning. A Laboratory Manual, 2ed., Cold Spring HarborLaboratory, Cold Spring Harbor, 1989. Further examples of stringentconditions can be found in Goeddel et al., U.S. Pat. No. 5,789,550.

In a non-limiting example, “stringent conditions” can be provided in avariety of ways such as overnight incubation at 42° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.Alternatively, the stringent conditions are characterized by ahybridization buffer comprising 30% formamide in 5×SSPE (0.18 M NaCl,0.01 M NaPO₄, pH 7.7, 0.0001 M EDTA) buffer at a temperature of 42° C.,and subsequent washing at 42° C. with 0.2×SSPE. Preferably, stringentconditions involve the use of a hybridization buffer comprising 50%formamide in 5×SSPE at a temperature of 42° C. and washing at the sametemperature with 0.2×SSPE.

As used herein, by “preserves progenitor cells” is meant an ability of aFRIL family member molecule (or mutant thereof or fusion proteincomprising a FRIL family member molecule or mutant thereof) to retain(i.e., preserve) progenitor cells in an undifferentiated state, whichcan be determined using the assays described below (e.g., the SCID mousereconstituting cell assay and the methylcellulose or other semi-solidmedium based hematopoietic progenitor cell assay). In accordance withthe invention, “progenitor cell” refers to any normal somatic cell thathas the capacity to generate fully differentiated, functional progeny bydifferentiation and proliferation. Progenitor cells include progenitorsfrom any tissue or organ system, including, but not limited to, blood,mesenchymal, embryonic, nerve, muscle, skin, gut, bone, kidney, liver,pancreas, thymus, brain and the like. Progenitor cells are distinguishedfrom “differentiated cells,” the latter being defined as those cellsthat may or may not have the capacity to proliferate, i.e.,self-replicate, but that are unable to undergo further differentiationto a different cell type under normal physiological conditions.Moreover, progenitor cells are further distinguished from abnormal cellssuch as neoplastic cells, as defined herein. For example, leukemia cellsproliferate (self-replicate), but generally do not furtherdifferentiate, despite appearing to be immature or undifferentiated.

Progenitor cells include all the cells in a lineage of differentiationand proliferation prior to the most differentiated or the fully maturecell. Thus, for example, progenitors include the skin progenitor in themature individual. The skin progenitor is capable of differentiation toonly one type of cell, but is itself not fully mature or fullydifferentiated.

By “hematopoiesis” is meant the development of mature, functional bloodcells. The progenitor cells that give rise to mature, functional bloodcells are called hematopoietic progenitor cells. The most primitive,undifferentiated hematopoietic progenitor cell is called a hematopoieticstem cell. Hematopoietic stem cells typically reside in the bone marrowprimarily in a quiescent state, and may form identical daughter cellsthrough a process called “self-renewal.”

Production of some mature, functional blood cells results fromproliferation and differentiation of “unipotential progenitors,” i.e.,those progenitors that have the capacity to make only one type of bloodcell. For red blood cell (erythrocyte) production, a unipotentialprogenitor called a “CFU-E” (colony forming unit-erythroid) has thecapacity to generate two to 32 mature progeny cells. Various otherhematopoietic progenitors have been characterized. For example,hematopoietic progenitor cells include those cells that are capable ofsuccessive cycles of differentiating and proliferating to yield up toeight different mature hematopoietic cell lineages.

Uncommitted progenitor cells, such as hematopoietic stem cells, can bedescribed as being “totipotent,” i.e., both necessary and sufficient forgenerating all types of mature cells. Progenitor cells that retain acapacity to generate all cell lineages, but that can not self-renew, aretermed “pluripotent.” Cells that can produce some but not all bloodlineages and can not self-renew are termed “multipotent.”

Progenitor cells can be defined by mRNA levels of genes that eitherspecifically regulate progenitors or serve as markers of lineagecommitment. For example, genes induced in primitive human hematopoieticprogenitor cells include those encoding the shared beta subunits of theIL3, IL5, and/or granulocyte-macrophage colony-stimulating factor(GM-CSF) receptors, termed the beta common chain (McClanahan et al.,Blood 81:3903-2915, 1993); CD34 genes; and/or the receptors for Kit(Turner et al., Blood 88:3383-3390, 1996), FLT1, FLT4 (Galland et al.,Oncogene 8:3233-1240, 1993), FLK1 (Broxmeyer et al., Int. J. Hematol.62:303-215, 1995), and FLT3 (Lyman and Jacobsen, Blood 91:3101-1134.,1998). Those genes for intermediate progenitors include the c-Fms, G-CSFreceptor, and/or CD34 genes; and the IL-7 receptor gene, a gene inducedfor B lymphopoiesis.

Murine primitive progenitor populations include receptors forinterleukin-1 alpha (IL-1α), IL-3, IL-6, granulocyte colony-stimulatingfactor (G-CSF), and/or FLK1-1 (the murine homologue of human KDR whichbinds VEGF) (Broxmeyer, supra), but lack receptors for macrophagecolony-stimulating factor (M-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Cellswithin the intermediate progenitor cell population include receptors forGM-CSF, G-CSF, IL-6, and/or IL-1α.

In accordance with the first aspect of the invention, the terms “bind,”“binds,” or “bound” are used interchangeably to mean that a FRIL familymember molecule of the invention binds to a normally glycosylated FLT3receptor with an affinity higher than the affinity with which theFLT3-Ligand binds the FLT3 receptor. Preferably, a FRIL family membermolecule binds to a normally glycosylated FLT3 receptor with an affinitythat is at least as high as the affinity with which an antibody bindsits specific ligand. Even more preferably, a FRIL family member moleculeof the invention binds to a normally glycosylated FLT3 receptor with anaffinity that is higher than the affinity with which an antibody bindsits specific ligand. Still more preferably, a FRIL family membermolecule of the invention binds to a normally glycosylated FLT3 receptorwith a dissociation constant (K_(D)) of at least 10⁻⁷ M, more preferably10⁻⁸ M, even more preferably 10⁻⁹ M, still more preferably, at least10⁻¹⁰ M, and most preferably, a FRIL family member molecule of theinvention binds to a normally glycosylated FLT3 receptor with adissociation constant (K_(D)) of at least 10⁻¹¹ M. Standard methods fordetermining binding and binding affinity are known.

In accordance with the invention, by “normally glycosylated FLT3receptor” is meant an FLT3 receptor that has a glycosylation pattern ofan FLT3 cell glycosylated by a normal cell. By “normal cell,” as usedherein in accordance with all aspects of the present invention, is meanta cell that is not neoplastic. As used herein, by “neoplastic cell” ismeant a cell that shows aberrant proliferation, particularly increasedproliferation, that is not regulated by such factors as cell-cellcontact inhibition and soluble regulators (e.g., cytokines or hormones),and that abnormally glycosylates the FLT3 receptor such that theglycosylation pattern on the FLT3 receptor on the neoplastic cells isabnormal and such that the FLT3 receptor on the neoplastic cell is notbound by a FRIL family member molecule.

In accordance with the first aspect of the invention, by “essentiallypure” means a molecule, such as a nucleic acid or protein (e.g., a FRILfamily member molecule), or composition of a molecule that is more freefrom other organic molecules (e.g., carbohydrates, nucleic acids,proteins, and lipids) that naturally occur with an impure molecule, andis substantially free as well of materials used during the purificationprocess. For example, a protein or nucleic acid molecule is consideredto be essentially pure if it is at least approximately 60%, preferablyat least approximately 75%, more preferably approximately at least 85%,most preferably approximately at least 90%, and optimally approximatelyat least 95% pure, i.e., free from other organic molecules with which itnaturally occurs and free from materials used during the purificationprocess. Methods for purifying proteins are known in the art andinclude, without limitation, HPLC, SDS-PAGE, immunoprecipitation,recombinant protein production, affinity chromatography using specificantibodies, ion-exchange, size-exclusion, and hydrophobic interactionchromatography, or a combination of any of these methods. These andother suitable methods are described, e.g., in Marston, “Thepurification of eukaryotic proteins expressed in E. coli,” in DNACloning, Glover D. M., ed., Volume III, IRL Press Ltd., Oxford, 1987;Marston and Hartley, “Solubilization of protein aggregates,” pp. 266-267in Guide to Protein Purification, Deutscher M P, ed., Academic Press,San Diego, 1990; Laemmli, U. K., Nature 227:680-685, 1970. A FRIL familymember can also be purified by binding to a mannose, which may becoupled on a sold support (e.g., a sepharose bead).

Methods for purifying nucleic adds are known in the art and include,without limitation, Guanidine-HCl extraction, polymerase chain reaction,CsCl gradient fractionation, phenol: chloroform extraction, ethanolprecipitation, and standard recombinant DNA methodologies. Standardmethods for purifying both proteins and nucleic acid molecules areprovided in, e.g., Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1994; Sambrook et al.,supra.

In accordance with the first aspect of the invention, a FRIL familymember molecule may be purified from a natural source by methods wellknown in the art. For example, the purification of Dl-FRIL from Dolichoslab lab is described below in Example 1. The purification of Pv-FRILfrom Phaseolus vulgaris as described below in Example 5. Thepurification of YamFRIL from Sphenostylis stenocarpa is described belowin Example 22. Such methods also include, for example, those describedby Moore in PCT application PCT/US97/22486 and by Gowda et al., supra. Asuitable natural source from which to purify a FRIL family membermolecule includes plants, especially legume plants. Legumes, such as thegarden pea or the common bean, are plants (“leguminous plants”) from afamily (Leguminosae) of dicotyledonous herbs, shrubs, and trees bearing(nitrogen-fixing bacteria) nodules on their roots. These plants arecommonly associated with their seeds (e.g., the garden pea or the commonbean)

More specifically, a FRIL family member molecule according to the firstaspect of the invention can be purified from members of the tribePhaseoleae. For example, a FRIL family member molecule can be purifiedfrom Dolichos lab lab (e.g., hyacinth beans, which is also known byother common names throughout the world). Alternatively, a FRIL familymember molecule can be purified from varieties of the common bean(Phaseolus vulgaris) (e.g., red kidney beans and white kidney beans),from yam bean (Sphenostylis stenocarpa) or from Vigna sinensis, commonlyknown as the black-eyed pea.

As demonstrated in the examples below, purification of a FRIL familymember molecule from a legume is rapid and inexpensive, and results in alarge amount of essentially pure lectin. A native FRIL family membermolecule can be easily purified from legumes, such as hyacinth beans(pesticide-free), by mannose-affinity chromatography or ovalbuminaffinity chromatography, and is more than 100 times cheaper to producethan recombinant cytokines. In accordance with the first aspect of theinvention, by “lectin” is meant a protein that binds sugar residues withhigh affinity. Most preferably, a FRIL family member molecule is amannose/glucose-specific legume lectin.

As demonstrated in the examples below, FRIL family member molecules, andcompositions of FRIL family member molecules, have many attributes asreagents to either alleviate the progenitor cell-depleting activity of atherapeutic (e.g., a chemotherapeutic) or to alleviate the symptoms of acondition where the patient's progenitor cells are depleted. Forexample, FRIL family members have unique properties and are the firstsoluble regulators reported to preserve hematopoietic stem cells andprogenitors in a dormant state for extended periods, even in thepresence of potent stimulators of proliferation and differentiation.Moreover, because mice tolerate very high levels of compositions of FRILfamily members, this may permit more effective protection of stem cellsand progenitors by preventing their recruitment during aggressive doseintensification regimens aimed at increasing frequency and dosage levelsof chemotherapy. While the biological activity of Dl-FRIL is similar tocytokines (ng/ml range), as demonstrated in the examples below, micetolerated up to a 1,000-fold more Dl-FRIL than cytokines.

In addition, by preserving hematopoietic stem cells and other progenitorcells in a dormant state, one or more members of the FRIL family allowsfor the administration of a broad range of cell cycle activechemotherapy drugs with greater frequency and higher dose. Thus,administration of a composition of one or more FRIL family members maypermit more aggressive dose-intensification chemotherapy regimens for abroad range of chemotherapy drugs. Administration of a composition of aFRIL family member also provides for a larger reservoir of progenitorcells which could rapidly respond to stimulatory signals aftercompleting chemotherapy.

In certain embodiment of the first aspect of the invention, the FRILfamily member of the invention is from a legume (e.g., a bean plant).

In some embodiments, the FRIL family member molecule of the invention isfrom a hyacinth bean (i.e., Dolichos lab lab), and has an amino addsequence comprising the sequence of SEQ ID NO: 24. Preferably, the FRILfamily member molecule of the invention isolated from a hyacinth beanhas the amino add sequence of SEQ ID NO: 2 or comprises a signalsequence having the amino add sequence of SEQ ID NO: 4, and, even morepreferably, is encoded by a nucleic add having the nucleic add sequenceof SEQ ID NO: 1 or the nucleic add sequence of SEQ ID NO: 3.

In some embodiments, the FRIL family member molecule of the invention isfrom a red kidney bean (i.e., Phaseolus vulgaris). Preferably, the FRILfamily member of the invention isolated from a red kidney bean has theamino acid sequence of SEQ ID NO: 6, and, even more preferably, isencoded by a nucleic add having the nucleic add sequence of SEQ ID NO:5.

In some embodiments, the FRIL family member of the invention is from ayam bean (i.e., Sphenostylis stenocarpa). Preferably, the FRIL familymember of the invention isolated from a yam bean has the amino acidsequence comprising the amino acid sequence of SEQ ID NO: 8, morepreferably has a β subunit having an amino acid sequence which comprisesthe amino acid sequence of SEQ ID NO: 9, even more preferably has an αsubunit having an amino acid sequence which comprises the amino acidsequence of SEQ ID NO: 10, and, even more preferably, is encoded by anucleic acid having a nucleic acid sequence which comprises the nucleicacid sequence of SEQ ID NO: 7.

In certain embodiments of the first aspect of the invention, the FRILfamily member molecule is a mutant derived from a second member of theFRIL family, wherein the mutant is selected from the group consisting ofa substitution mutant, a deletion mutant, an addition mutant, or acombination thereof (e.g., a mutant of Dl-FRIL or Pv-FRIL describedbelow). For example, it is preferred to substitute amino acids in asequence with equivalent amino acids. Groups of amino acids knownnormally to be equivalent are: (1) Ala(A), Ser(S), Thr(T), Pro(P), andGly(G); (2) Asn(N), Asp(D), Glu(E), Gln(Q); (3) His(H), Arg(R), Lys(K);(4) Met(M), Leu(L), Ile(I), Val(V); and (5) Phe(F), Tyr(Y), Trp(W).Substitutions, additions, and/or deletions in an amino acid sequence canbe made as long as the mutant FRIL family member molecule continues tosatisfy the functional criteria described herein. An amino acid sequencethat is substantially the same as another sequence, but that differsfrom the other sequence by means of one or more substitutions,additions, and/or deletions, is considered to be an equivalent sequence.Preferably, less than 50%, more preferably less than 25%, and still morepreferably less than 10%, of the number of amino acid residues in asequence are substituted for, added to, or deleted from the FRIL familymember molecule upon which the mutant FRIL family member was derived.

In certain embodiments of the first aspect of the invention, the FRILfamily member molecule is a fusion protein comprising a first portionand a second portion, wherein the first portion is derived from a secondmember of the FRIL family. By “fusion protein” is meant a moleculecomprising at least two proteins or polypeptide fragments thereof joinedtogether, wherein the proteins or polypeptide fragments thereof are notjoined together in the naturally-occurring organism from which theproteins or polypeptide fragments thereof were derived. The two proteinsor polypeptide fragments thereof of a fusion protein may be joined byany means, including, without limitation, a chemical linker, a peptidebond, or a non-covalent bond, such as an ionic bond. By “protein” or“polypeptide” is meant a chain of two or more amino acid residues joinedwith a peptide bond regardless of length or post-translationalmodification such as acetylation, glycosylation, lipidation,acetylation, or phosphorylation.

A FRIL family member molecule that is a fusion protein may comprise afirst portion derived from a FRIL family member and a second portionderived from a protein or other molecule not related to the FRIL family(eg., the heavy chain of an antibody).

An additional FRIL family member molecule that is a fusion protein isFRIL family member comprising the α subunit from a first FRIL familymember and a β subunit from a second FRIL family member. Such a fusionprotein may be generated, for example, by joining a nucleic acidsequence encoding the α subunit of the first FRIL family member in framewith a nucleic acid sequence encoding the β subunit of the second FRILfamily member. The nucleic add encoding such a fusion protein can beengineered to encode an enzyme-specific cleavage site between theportion encoding the α subunit of the first FRIL family member and theportion encoding the β subunit of the second FRIL family member.

Where a FRIL family member is a fusion protein, identity of the fusionprotein as a FRIL family member is determined by the sequence identitybetween the FRIL family member-derived portion of the fusion protein anda second FRIL family member, where the FRIL family member-derivedportion of the fusion protein and the second FRIL family member share atleast about 45% amino acid sequence identity, even more preferably atleast about 50% identity, even more preferably at least about 55%identity, still more preferably at least about 60% identity, still morepreferably at least about 65% identity yet more preferably at leastabout 75% sequence identity, still more preferably at least about 85%identity, and most preferably at least about 95% identity, with theamino acid sequence of a second member of the FRIL family.

A FRIL family member in accordance with the first aspect of theinvention can also be a recombinant protein made by expressing arecombinant nucleic acid that encodes FRIL in a suitable host. Thus, ina second aspect, the invention features an essentially pure nucleic acidmolecule encoding a member of the FRIL family of progenitor cellpreservation factors. Exemplary purifications of the nucleic acidmolecules of the invention from Dolichos lab lab and Phaseolus vulgarisare described below. As is well known, if an amino acid sequence(primary structure) is known, a family of nucleic acids can then beconstructed, each having a sequence that differs from the others by atleast one nucleotide, but where each different nucleic acid stillencodes the same protein. For example, if a protein has been sequencedbut its corresponding gene has not been identified, the gene can beacquired through amplification of genomic DNA using a set of degenerateprimers that specify all possible sequences encoding the protein. Thus,a nucleic acid in accordance to this aspect of the invention need nothave a naturally occurring sequence, but need only encode a FRIL familymember according to the first aspect of the invention.

In accordance with the second aspect of the invention, a “member of theFRIL family of progenitor cell preservation factors” is a describedabove the first aspect of the invention. “Essentially pure” is used asdescribed for the first aspect of the invention.

By a “recombinant nucleic acid” is meant a nucleic acid which encodes aFRIL family member molecule, or a portion encoding at least 15contiguous amino acids thereof, or a mutant thereof, or a fusion proteincomprising the molecule, portion thereof or mutant thereof or is capableof expressing an antisense molecule specifically complementary thereto,or a sense molecule that shares nucleic acid sequence identity theretowherein the recombinant nucleic acid may be in the form of linear DNA orRNA, covalently closed circular DNA or RNA, or as part of a chromosome,provided however that it cannot be the native chromosomal locus for aFRIL family member molecule. Preferred recombinant nucleic acids of theinvention are vectors, which may include an origin of replication andare thus replicatable in one or more cell type. Certain preferredrecombinant nucleic acids are expression vectors, and further compriseat least a promoter and passive terminator, thereby allowingtranscription of the recombinant nucleic acid in a bacterial, fungal,plant, insect or mammalian cell. By “nucleic acid” or “nucleic acidmolecule” as used herein, means any deoxyribonucleic acid (DNA) orribonucleic acid (RNA), including, without limitation, complementary DNA(cDNA), genomic DNA, RNA, hnRNA, messenger RNA (mRNA), DNA/RNA hybrids,or synthetic nucleic acids (e.g., an oligonucleotide) comprisingribonucleic and/or deoxyribonucleic acids or synthetic variants thereof.The nucleic acid of the invention includes, without limitation, anoligonucleotide or a polynucleotide. The nucleic acid can be singlestranded, or partially or completely double stranded (duplex). Duplexnucleic acids can be homoduplex or heteroduplex.

In accordance with the second aspect of the invention, a nucleic acidencoding a FRIL family member has at least about 50% nudeic acidsequence identity with a nucleic acid encoding another member of theFRIL family, preferably at least about 555% nucleic acid sequenceidentity, more preferably at least about 60% nucleic acid sequenceidentity, more preferably at least about 65% nucleic acid sequenceidentity, still more preferably at least about 75% nudeic acid sequenceidentity, still more preferably at least about 85% nucleic acid sequenceidentity, and most preferably at least about 95% nucleic acid sequenceidentity with a nucleic acid encoding another member of the FRIL family.Percentage nudeic acid sequence identity can be determined as describedfor the first aspect of the invention.

A recombinant nucleic acid according to the second aspect of theinvention can also be chemically synthesized by methods known in theart. For example, recombinant DNA can be synthesized chemically from thefour nucleotides in whole or in part by methods known in the art. Suchmethods include those described in Caruthers, M. H., Science230(4723):281-285, 1985). DNA can also be synthesized by preparingoverlapping double-stranded oligonucleotides, filling in the gaps, andligating the ends together. See, generally, Sambrook et al., supra, andGlover and Hames, eds., DNA Cloning, 2d ed., Vols. 14, IRL Press,Oxford, 1995.

A recombinant nucleic acid molecule of the invention encoding a mutantFRIL family member can be prepared from wild-type DNA by site-directedmutagenesis (see, for example, Zoller and Smith, Nucleic. Acids. Res.10:6487-6500, 1982; Zoller, M. J., Methods Enzymol. 100:468-500, 1983;Zoller, M. J., DNA 3(6):479-488, 1984.; and McPherson, M. J., ed.,Directed Mutagenesis: A Practical Approach, IRL Press, Oxford, 1991.

A recombinant nucleic acid of the second aspect of the invention can beamplified by methods known in the art. One suitable method is thepolymerase chain reaction (PCR) method described in Saiki et al.,Science 239:487, 1988, Mullis et al., U.S. Pat. No. 4,683,195, andSambrook et al., supra. It is convenient to amplify the clones in thelambda-gt10 or lambda-gt11 vectors using lambda-gt10- orlambda-gt11-specific oligomers as the amplimers (available fromClontech, Palo Alto, Calif.). Larger synthetic nucleic acid structurescan also be manufactured having specific and recognizable utilitiesaccording to the invention. For example, vectors (e.g., recombinantexpression vectors) are known which permit the incorporation ofrecombinant nucleic acids of interest for cloning and transformation ofother cells. Thus, the invention further includes vectors (e.g.,plasmids, phages, and cosmids) which incorporate a nucleotide sequenceof the invention, especially vectors which include the recombinantnucleic acid molecule of the invention for expression of a FRIL familymember.

A recombinant nucleic acid of the invention can be replicated and usedto express a FRIL family member following insertion into a wide varietyof host cells in a wide variety of cloning and expression vectors. Thehost can be prokaryotic or eukaryotic. The nucleic acid can be obtainedfrom natural sources and, optionally, modified. The genes can also besynthesized in whole or in part.

Cloning vectors can comprise segments of chromosomal, non-chromosomaland synthetic DNA sequences. Some suitable prokaryotic cloning vectorsinclude plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC,pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNAsuch as M13fd, and other filamentous single-stranded DNA phages.

Vectors for expressing proteins in bacteria, especially E. coli, arealso known. Such vectors include the pK233 (or any of the tac family ofplasmids), T7, and lambda P_(L). Examples of vectors that express fusionproteins are PATH vectors described in Dieckmann and Tzagoloff (J. Biol.Chem. 260(3):1513-1520, 1985). These vectors contain DNA sequences thatencode anthranilate synthetase (TrpE) followed by a polylinker at thecarboxy terminus. Other expression vector systems are based onbeta-galactosidase (pEX); maltose binding protein (pMAL); glutathioneS-transferase (pGST) (see, e.g., Smith, D. B., Gene 67:31-40, 1988 andAbath, F. G., Peptide Research 3(4):167-168, 1990). Vectors useful forcloning and expression in yeast are also available. A suitable exampleis the 2 μ circle plasmid.

Suitable cloning/expression vectors for use in mammalian cells are alsoknown. Such vectors include well-known derivatives of SV-40, adenovirus,cytomegalovirus (CMV) retrovirus-derived DNA sequences. Any suchvectors, when coupled with vectors derived from a combination ofplasmids and phage DNA, i.e., shuttle vectors, allow for the isolationand identification of protein coding sequences in prokaryotes.

Further eukaryotic expression vectors are known in the art (e.g.,Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982; Subramani etal., Mol. Cell. Biol. 1:854-864, 1981; Kaufmann and Sharp, J. Mol. Biol.159:601-621, 1982; Kaufmann and Sharp, Mol. Cell. Biol. 159:601-664,1982; Scahill et al., Proc. Natl. Acad. Sci. USA 80:4654-4659, 1983;Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).

The expression vectors useful in the present invention contain at leastone expression control sequence that is operatively linked to therecombinant nucleic acid molecule or fragment thereof to be expressed.The control sequence is inserted in the vector in order to control andto regulate the expression of the recombinant nucleic acid of theinvention. Examples of useful expression control sequences are the lacsystem, the trp system, the tac system, the trc system, major operatorand promoter regions of phage lambda, the control region of fd coatprotein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof.

Useful expression hosts for expressing the recombinant nucleic acids ofthe invention include well-known prokaryotic and eukaryotic cells. Somesuitable prokaryotic hosts include, for example, E. coli, such as E.coli SG-936, E. coli HB101, E. coli W3110, E. coli X1776, E. coli X2282,E. coli DHI, and E. coli MRCl, Pseudomonas, Bacillus, such as B.subtilis, and Streptomyces. Suitable eukaryotic cells include yeasts andother fungi, insect, animal cells, such as COS cells and CHO cells,human cells and plant cells in tissue culture.

Given the recombinant nucleic acid sequences disclosed herein, theartisan can further design recombinant nucleic acids having particularfunctions in various types of applications. For example, the artisan canconstruct oligonucleotides or polynucleotides for use as primers innucleic acid amplification procedures, such as the polymerase chainreaction (PCR), ligase chain reaction (LCR), Repair Chain Reaction(RCR), PCR oligonucleotide ligation assay (PCR-OLA), and the like.Oligonucleotides useful as probes in hybridization studies, such as insitu hybridization, can be constructed. Numerous methods for labelingsuch probes with radioisotopes, fluorescent tags, enzymes, bindingmoieties (e.g., biotin), and the like are known, so that the probes ofthe invention can be adapted for easy detectability.

Oligonucleotides can also be designed and manufactured for otherpurposes. For example, the invention enables the artisan to designantisense oligonucleotides, and triplex-forming oligonucleotides, andthe like, for use in the study of structure/function relationships.Homologous recombination can be implemented by adaptation of the nucleicacid of the invention for use as targeting means.

Recombinant nucleic acids of the invention produced as described abovecan further be modified to alter biophysical or biological properties bymeans of techniques known in the art. For example, the recombinantnucleic acid can be modified to increase its stability against nucleases(e.g., “end-capping”), or to modify its lipophilicity, solubility, orbinding affinity to complementary sequences. Methods for modifyingnucleic acids to achieve specific purposes are disclosed in the art, forexample, in Sambrook et al., supra. Moreover, the recombinant nucleicacid of the invention can include one or more portions of nucleotidesequence that are non-coding for a FRIL family member.

In a third aspect, the invention provides a pharmaceutical formulationcomprising an essentially pure composition of one or more members of theFRIL family of progenitor cell preservation factors and apharmaceutically acceptable carrier. By “pharmaceutically acceptablecarrier” is meant any inert carrier that is non-toxic to the animal towhich it is administered and that retains the therapeutic properties ofthe compound with which it is administered (i.e., the FRIL familymember). Pharmaceutically acceptable carriers and their formulations arewell-known and generally described in, for example, Remington'sPharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990). One exemplary pharmaceutically acceptablecarrier is physiological saline. Pharmaceutical formulations of theinvention may employ any pharmaceutically acceptable carrier, dependingupon the route of administration of the composition.

Compositions of FRIL family members may be used safely and efficaciouslyas a therapeutics. The gastrointestinal tracts of animals come inconstant contact with lectins, such as FRIL family members, in rawand/or cooked vegetables and fruits. Many lectins pass through thegastrointestinal tract biologically intact (Pusztai, A., Eur. J. Clin.Nutr. 47: 691-699, 1993). Some lectins interact with the gut and aretransported into the peripheral blood circulation. For example, a recentstudy found peanut agglutinin (PNA) in the blood of humans at levels of1-5 μg/ml an hour after ingesting 200 g of raw peanuts (Wang et al.,Lancet 352: 1831-1832, 1998). Antibodies to dietary lectins are commonlyfound in people at levels of ˜1 μg/ml (Tchernychev and Wilchek, FEBSLett. 397: 139-142, 1996). These circulating antibodies do not blockcarbohydrate binding of the lectins.

In certain embodiments of the third aspect of the invention,administration of a therapeutically effective amount of thepharmaceutical formulation to a patient suffering from a conditionwhereby the patient's hematopoietic progenitor cells are depletedalleviates and/or reduces the condition in the patient.

In accordance with the third aspect of the invention, by“therapeutically effective amount” is meant a dosage of a composition ofa FRIL family member or pharmaceutical formulation comprising acomposition of a FRIL family member that is effective to alleviateand/or reduce either a condition whereby the patient's hematopoieticprogenitor cells are depleted or a hematopoietic progenitorcell-depleting activity of a therapeutic (e.g., a chemotherapeutic).Preferably, such administration is systematic (e.g., by intravenousinjection). When administered systemically, a therapeutically effectiveamount is an amount of between about 500 ng of the FRIL family member/kgtotal body weight and about 5 mg/kg total body weight per day.Preferably, a therapeutically effective amount is between about 500ng/kg and 500 μg/kg total body weight of the FRIL family member per day.Still more preferably, a therapeutically effective amount is betweenabout 5 μg/kg and 50 μg/kg total body weight of the FRIL family memberper day. Most preferably, a therapeutically effective amount is anamount that delivers about 50 μg/kg total body weight of the FRIL familymember per day.

A composition of a FRIL family member of the invention andpharmaceutical formulation comprising a composition of a FRIL familymember of the invention may be administered patients having, orpredisposed to developing, a condition whereby the patient'shematopoietic progenitor cells are depleted. Such a condition may becongenital. For example, the patient may have severe combinedimmunodeficiency or aplastic anemia.

The condition may also be induced by a drug. Thus, in certainembodiments of the third aspect of the invention, administration of atherapeutically effective amount of the pharmaceutical formulation to apatient prior to treatment of the patient with a therapeutic treatmenthaving a hematopoietic progenitor cell-depleting activity alleviates thehematopoietic progenitor cell-depleting activity of the therapeutic inthe patient. For example, cancer patients are often treated withradiotherapeutics or chemotherapeutics that have hematopoieticprogenitor cell-depleting activity. By “hematopoietic progenitorcell-depleting activity” is meant an activity of a therapeutic treatmentwhereby the hematopoietic progenitor cells in the patient being treatedwith the therapeutic treatment are depleted, either by killing theprogenitor cells or by inducing the progenitor cells to undergoirreversible differentiation. Non-limiting examples of therapeutictreatments having hematopoietic progenitor cell-depleting activity arethe chemotherapeutic agents cytarabine (Ara-C), doxorubicin (Dox),daunorubicin, and 5-fluorouracil (5-FU).

In certain embodiments, administration of the pharmaceutical formulationof the invention to a patient prior to the treatment of the patient witha therapeutic treatment having a hematopoietic progenitor cell-depletingactivity enables treatment of the patient with a higher dosage of thetherapeutic treatment. The higher dosage of the therapeutic treatmentmay be accomplished by either an increased dose of the therapeutictreatment and/or an increased duration of treatment with the therapeutictreatment. For example, a child diagnosed with childhood AcuteMyelogenous Leukemia (AML) is typically initially treated for the firstseven days with daunorubicin at 45 mg/m² on Days 1-3 plus Ara-C at 100mg/m² for 7 days plus GTG at 100 mg/m² for 7 days. The same childpretreated with a composition in accordance with this aspect of theinvention may be able to tolerate a higher dosage (i.e., higher doseand/or prolonged treatment period) of any or all of thesechemotherapeutics. Such an increase in dosage tolerance of a therapeutictreatment(e.g., a chemotherapeutic) having a hematopoietic progenitorcell-depleting activity in a cancer patient is desirable since a higherdosage may result in the destruction of more cancerous cells.

The pharmaceutical formulations and/or compositions of the invention maybe administered by any appropriate means. For example, thepharmaceutical formulations and/or compositions of the invention may beadministered to an mammal within a pharmaceutically-acceptable diluent,carrier, or excipient, in unit dosage form according to conventionalpharmaceutical practice. Administration may begin before the mammal issymptomatic for a condition whereby the patient's hematopoieticprogenitor cells are depleted. For example, administration of thepharmaceutical formulations of the third aspect of the invention to acancer patient may begin before the patient receives radiotherapy and/orchemotherapy treatment.

Any appropriate route of administration of a pharmaceutical formulationand/or composition of the invention may be employed, including, withoutlimitation, parenteral intravenous, intra-arterial, subcutaneous,sublingual, transdermal, topical, intrapulmonary, intramuscular,intraperitoneal, by inhalation, intranasal, aerosol, intrarectal,intravaginal, or by oral administration. Pharmaceutical formulationsand/or compositions of the invention may be in the form of liquidsolutions or suspensions; for oral administration, formulations may bein the form of tablets or capsules; and for intranasal formulations, inthe form of powders, nasal drops, or aerosols. The pharmaceuticalformulations and/or compositions may be administered locally to the areaaffected by a condition whereby the patient's hematopoietic progenitorcells are depleted. For example, the pharmaceutical formulations and/orcompositions of the invention may be administered directly into thepatient's bone marrow. The pharmaceutical formulations and/orcompositions of the invention may be administered systemically.

In certain embodiments of the third aspect of the invention, the patientis human or a domesticated animal. By “domesticated animal” is meant ananimal domesticated by humans, including, without limitation, a cat,dog, elephants, horse, sheep, cow, pig, and goat. In some embodiments,the patient has cancer.

In some embodiments of the third aspect, the treatment is aradiotherapeutic or a chemotherapeutic treatment, including, withoutlimitation, cytarabine (Ara-C), doxorubicin (Dox), or 5-fluorouracil(5-FU), or a combination of a radiotherapeutic and a chemotherapeutic.

In a fourth aspect, the invention provides a method for a method foralleviating and/or reducing the hematopoietic progenitor cell-depletingactivity of a therapeutic treatment in a patient, comprisingadministering to the animal a therapeutically effective amount of acomposition of a FRIL family member prior to administration of thetherapeutic treatment to the patient. “Hematopoietic progenitorcell-depleting activity” is as described for the third aspect of theinvention. Routes of administration of a composition of a FRIL familymember of this aspect of the invention are as described for theadministration of the pharmaceutical formulation of the third aspect ofthe invention. “Therapeutically effective amount” is as described forthe third aspect of the invention.

In certain embodiments of the fourth aspect, the patient is a human or adomesticated animal. “Domesticated animal” is as described for the thirdaspect of the invention. In certain embodiments, the patient has cancer.In some embodiments, the therapeutic treatment is a radiotherapeutic ora chemotherapeutic treatment, including, without limitation, cytarabine(Ara-C), doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combinationof a radiotherapeutic and a chemotherapeutic.

In a fifth aspect, the invention provides a method for isolating apopulation of progenitor cells, comprising contacting a population ofcells with a plurality of FRIL family member molecules, and separatingthe unbound cells, wherein the cells bound to the FRIL family membermolecules are an isolated population of progenitor cells. “FRIL familymember molecule” and “progenitor cell” are as described for the firstaspect of the invention. By “unbound cell” is meant a cell that does notbind to a FRIL family member. “Bind” is a described for the first aspectof the invention.

By “isolated” is meant a population of progenitor cells that isseparated from a larger population of cells, wherein the percentage ofprogenitor cells in the isolated population is at least two fold greaterthan the percentage of progenitor cells in the larger population.Preferably, the percentage of progenitor cells in the isolatedpopulation is at least four fold greater than the percentage ofprogenitor cells in the larger population. A non-limiting example of theincreased percentage of progenitor cells in an isolated population ofprogenitor cells of the invention is shown below in Table 11.Preferably, the isolated population of progenitor cells of the inventionis at most 2% of the total population of umbilical cord blood mononudearcells (CB mnc). Preferably, the population of progenitor cells of theinvention is at most 1% of the total population of umbilical cord bloodmononuclear cells (CB mnc). Preferably, an isolated population ofprogenitor cells binds to a normally glycosylated FLT3 receptor.“Normally glycosylated FLT3 receptor” is as defined above.

In preferred embodiments, the isolated population of progenitor cells isfrom a human or is from a domesticated animal.

In certain embodiments of the fifth aspect of the invention, the FRILfamily member molecules are detectably labeled. By “detectably labeled”is meant that the FRIL family member is attached to a label that isdetectable visually or instrumentally. Detectable labels such as enzymesand chromophoric molecules can be conjugated to the FRIL family membermolecules by means of coupling agents, such as dialdehydes,carbodiimides, and dimaleimides. Numerous methods of labeling proteinsare known in the art. The label can also be directly attached through afunctional group on the FRIL family member. Such a functional group maybe present on the FRIL family member molecule to be detectably labeled;alternatively, the FRIL family member molecules can be modified usingstandard techniques to contain a functional group. Some examples ofsuitable functional groups include, without limitation, amino, carboxyl,sulfhydryl, maleimide, isocyanate, isothiocyanate.

In certain embodiments, the detectable label is radioactive ornon-radioactive. Some examples of useful radioactive labels include ³²P,¹²⁵I, ¹³¹I, and ³H. Use of radioactive labels have been described inU.K. patent document 2,034,323, and U.S. Pat. Nos. 4,358,535, and4,302,204. Some examples of non-radioactive labels include enzymes,chromophores, atoms and molecules detectable by electron microscopy, andmetal ions detectable by their magnetic properties.

In certain embodiments of the fifth aspect of the invention, thedetectable label is an enzymatic label. Some useful enzymatic labelsinclude enzymes that cause a detectable change in a substrate. Someuseful enzymes and their substrates include, for example, horseradishperoxidase (pyrogallol and o-phenylenediamine), beta-galactosidase(fluorescein beta-D-galactopyranoside), and alkaline phosphatase(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The useof enzymatic labels have been described in U.K. 2,019,404, and EP63,879, each incorporated herein by reference, and by Rotman, Proc.Natl. Acad. Sci. USA 47:1981-1991, 1961.

In certain embodiments of this aspect of the invention, the detectablylabeled FRIL family member molecules are labeled by being specificallybound by an antibody that is detectably labeled.

In certain embodiments of this aspect of the invention, the FRIL familymember molecules are conjugated to a receptor (or ligand) and isdetectably labeled by binding the receptor (or ligand) with the ligand(or receptor), wherein the ligand (or receptor) is detectably labeled.Any of the known ligand-receptor combinations is suitable. Some suitableligand-receptor pairs include, for example, biotin-avidin or-streptavidin, and antibody-antigen. In certain embodiments,biotin-avidin combination is preferred.

In certain embodiments, the detectable label is a chromophore. Usefulchromophores include, for example, fluorescent, chemiluminescent, andbioluminescent molecules, as well as dyes. Some specific chromophoresuseful in the present invention include, for example, fluorescein,rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

In certain embodiments of this aspect of the invention, where the FRILfamily member molecules are detectably labeled to a chromophore, theunbound cells are separated by using a flow cytometry cell sorter tosort the population of cells contacted with the FRIL family membermolecules detectably labeled with a fluorescent marker.

In certain embodiments of the fifth aspect of the invention, the FRILfamily member molecules are immobilized on a solid support. “Solidsupport” includes any surface, including, without limitation, thesurface of a sepharose bead, a gel, a matrix, a magnetic bead, and aplastic surface (e.g., the bottom of a tissue culture dish or flask).

In some embodiments, the solid support is a bead, for example, amagnetic bead. In some embodiments, where the solid support is amagnetic bead, the unbound cells are separated by applying a magnet tothe population of cells contacted with the FRIL family member moleculesimmobilized on the magnetic bead. In further embodiments, the populationof cells bound to the FRIL family member molecules immobilized on amagnetic bead is rinsed with a physiologically acceptable solution whilethe magnet is applied. By “physiologically acceptable solution” is meantan inert solution, such as sterile saline solution or tissue culturemedium, which is non-toxic to the cells.

Methods for isolating cells that bind FRIL family member molecule-coatedmagnetic beads are described below in Example 16. Magnetic beads arecommerically available (e.g., from Dynabeads Tosylactivated, LakeSuccess, N.Y.; or from Miltenyi Biotec, Auburn, Calif.). Since the FRILfamily member is protein, it can be conjugated to a magnetic bead viaamino- or sulfhydryl-groups of the protein. A FRIL family membermolecule can also immobilized on magnetic beads by a biotin-strepavidininteraction.

Preferred magnetic beads are the MACS super-paramagnetic MicroBeads(from Miltenyi Biotec) which are extremely small, approximately 50 nm indiameter (MACS beads are about one million times smaller in volume thaneukaryotic cells). Because MACS beads react like magnetic antibodies,magnetic labeling is achieved within minutes. MACS MicroBeads form astable colloidal suspension and do not precipitate or aggregate inmagnetic fields. Because of their size and composition (iron oxide andpolysaccharide) make the MACS biodegradable, so labeled cells retaintheir physiological function. This property of MACS beads isparticularly useful for bead-sorted FRIL family member-binding cells,which bind the FRIL family member with such high affinity that it isdifficult to remove the beads.

In certain embodiments of the fifth aspect, the solid support is thebottom of a tissue culture plate. In some embodiments, the unbound cellsare separated by rinsing the population of cells contacted with the FRILfamily member molecules immobilized on the bottom of a tissue cultureplate with a physiologically acceptable solution.

FRIL family member molecules may be directly or indirectly attached tothe bottom of a tissue culture plate. Following a standard “panning”protocol (see, e.g., Stengelin et al., EMBO J. 7(4):1053-1059, 1988;Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84(23): 8573-8577, 1987), apopulation of cells suspected of containing FRIL family member-bindingprogenitor cells is incubated on the plate. The plate is then gentlyrinsed with a physiologically acceptable solution, thereby removing theunbound cells while leaving the FRIL family member-binding population ofcells attached to the FRIL family member-coated plate.

In preferred embodiments of the fifth aspect of the invention, theisolated population of progenitor cell is a population of hematopoieticprogenitor cells. In various embodiments, the population of cells iswhole blood, umbilical cord blood, bone marrow cells, or fetal livercells.

In certain embodiments of the fifth aspect of the invention, thepopulation of cells is a sorted population of cells, wherein a cell ofthe sorted population does not express CD11b, CD11c, or CD38. Becausemore primitive progenitor cell stypically expresses few cell surfacemolecules, prior to isolating a population of progenitor cells thatbinds to a FRIL family member, the population of cells is preferablysorted to first remove cells that express one or more of the followingcell surface molecules: CD11b, CD11c, and CD38. Following this negativesort (i.e., a sort, wherein the cells retained do not express CD11b,CD11c, and/or CD38), the sorted population is positively sorted for anability to bind a FRIL family member.

In certain embodiments, the sorted population of cells is sorted by flowcytometry or by magnetic bead selection. Thus, a population of cells(e.g., human umbilical cord blood cells) may be first contacted withchromophore-labeled antibodies (or other molecule such as a ligand)which specifically bind CD11b, CD11c, and/or CD38. Following binding,the population of cells is then negatively sorted by flow cytometry,where the cells which are not bound by the antibodies (and so do notexpress CD11b, CD11c, and/or CD38) are retained and further sorted foran ability to bind a FRIL family member, wherein the population of cellsthat binds a FRIL family member is an isolated population of progenitorcells according to the invention.

A population of cells may also be contacted with a molecule, such as anantibody, which specifically binds CD11b, CD11c, and/or CD38, whereinthe molecule is attached to a solid support, such as a magnetic bead.The population is then negatively sorted by applying a magnet to thebeads, and retaining the cells that do not bind the beads and so are notattracted to the magnet. These sorted cells are then further sorted foran ability to bind a FRIL family member, wherein the population of cellsthat binds a FRIL family member is an isolated population of progenitorcells according to the invention.

In a sixth aspect, invention provides an isolated population ofprogenitor cells isolated by a method comprising contacting a populationof cells with a plurality of FRIL family member molecules, andseparating the unbound cells, wherein the cells bound to the FRIL familymember are an isolated population of progenitor cells. “FRIL familymember molecule,” “progenitor cell,” “unbound cell,” and “bind” are asdescribed above.

In preferred embodiments, the isolated population of progenitor cells isfrom a human or a domesticated animal.

In certain embodiments of the sixth aspect, the cells of the isolatedpopulation do not express CD34 on their cell surface. In certainembodiments, the cells of the isolated population express the FLK1,FLT1, FLT3, FLT4, or Kit receptor tyrosine kinases. In some embodiments,the cells of the isolated population express the CD11b or CD11c cellsurface molecules. In preferred embodiments, the cells of the isolatedpopulation express FLT3.

In various embodiments of the sixth aspect of the invention, the cellsof the isolated population are hemangioblasts, messenchymal stem cells,bone progenitor cells, hepatic progenitor cells, endothelial progenitorcells, hematopoietic progenitor cells, embryonal stem cells, brainprogenitor cells, or dendritic progenitor cells. By “hemangioblast” ismeant a cell that is a progenitor cell for both hemaopoietic andendothelial lineages. Preferably, the cells of the isolated populationare hematopoietic progenitor cells. By a “messenchymal stem cells” ismeant the population of cells that is the progenitor for bone marrowstromal cells, including, without limitation, adipose tissue cells,cartilage-producing cells, muscle cells, and bone cells. Such cells ofthis aspect of the invention can be used, for example, for tissuerepair.

Determination of what type of cells a progenitor cell of the inventionwill give rise to is made by the various progenitor cell assaysdescribed below. For example, if a progenitor cell is suspected of beingan endothelial progenitor cell, the cell may be cultured in amethylcellulose progenitor cell assay in the presence of vascularendothelial growth factor. Should cells arising from such a culture haveendothelial cell markers, the progenitor cell of the invention is aendothelial progenitor cell or perhaps a hemangioblast.

In certain embodiments of the sixth aspect of the invention, where thecells of the isolated population are hematopoietic progenitor cells,transplantation of the cell into an animal lacking a population ofhematopoietic progenitor cells sufficient to enable survival of theanimal reconstitutes the animal, wherein the transplanted animalsurvives. Determination of the ability of a hematopoietic progenitorcell to reconstitute a animal lacking a population of hematopoieticprogenitor cells sufficient to enable survival of the animal may be madeusing the methods described below for the NOD-SCID mouse.

In certain embodiments, the hematopoietic progenitor cells are from amouse or a human and the animal is a mouse. In some embodiments, themouse is a severe combined immunodeficient (SCID) mouse (e.g., theNOD-SCID mouse described below) or a mouse that has been exposed to asublethal dose of radiation and/or chemotherapy

In certain embodiments of the sixth aspect of the invention, thehematopoietic progenitor cells are from a human and the animal is ahuman. In some embodiments, the human is a cancer patient receiving atreatment that depletes the patient's hematopoietic progenitor cells. Insome embodiments, the treatment is a radiotherapeutic or achemotherapeutic treatment, induding, without limitation, cytarabine(Ara-C), doxorubicin (Dox), or 5-fluorouracil (5-FU), or a combinationof a radiotherapeutic and a chemotherapeutic.

In a seventh aspect, the invention provides a method for preservingprogenitor cells ex vivo, comprising contacting a population of cellscomprising at least one progenitor cell with an effective amount of acomposition of a FRIL family member for an effective period of time,wherein the progenitor cell in the population are rendered quiescent.“FRIL family member” and “progenitor cell” are as described above forthe first aspect of the invention.

In accordance with this aspect of the invention, the terms “effectiveamount” and “effective period of time” are used to denote knowntreatments at dosages and for periods of time effective to preserveprogenitor cells. Where administered to a patient, preferably, suchadministration is systemic (e.g., by intravenous injection). Effectiveamounts and effective periods of time can be determined using the modelsand assays described herein. For example, the Examples below describethe preservation of progenitor cells that have SCID-reconstitutingability. In accordance with the invention, an effective amount may rangefrom about 0.1 ng/mL to about 1 μg/mL of a FRIL family member,preferably about 1.0 ng/mL to to about 1.0 μg/mL, more preferably about1.0 ng/mL to about 100 ng/mL, even more preferably about 10 ng/mL toabout 50 ng/mL, and most preferably about 50 ng/mL of a FRIL familymember in culture. In accordance with the invention, an effective periodof time includes culturing the cells in the presence of a FRIL familymember for between would include from about 2 hours to 5 days, morepreferably from about 12 hours to about 3 days, and most preferably forabout 24 hours.

In certain embodiments of the seventh aspect, the progenitor cells arefrom a human or from a domesticated animal. In certain embodiments, thepopulation of cells is bone marrow cells.

In some embodiments, the non-progenitor cells in the population of cellsdifferentiate or die. Thus, although the progenitor cells in the culturedo not actually expand in number, they are enriched relative to thenumber of cells in the culture.

In certain embodiments of the seventh aspect, the population of cells isremoved from a cancer patient prior to treatment of the cancer patientwith a therapeutic treatment having a hematopoietic progenitorcell-depleting activity. In some embodiments, the therapeutic treatmentis a radiotherapeutic or a chemotherapeutic treatment, including,without limitation, cytarabine (Ara-C), doxorubicin (Dox), or5-fluorouracil (5-FU), or a combination of a radiotherapeutic and achemotherapeutic.

In an eighth aspect, the invention provides a method for preservingprogenitor cells in vivo, comprising administering to a patient atherapeutically effective amount of a composition of a FRIL familymember for a therapeutically effective period of time, wherein theprogenitor cells in the patient are rendered quiescent. “FRIL familymember” and “progenitor cell” are as described above for the firstaspect of the invention. “Therapeutically effective amount” is asdescribed for the third aspect of the invention.

By “therapeutically effective period of time” is meant treatment for aperiod of time effective to preserve progenitor cells. Whereadministered to a patient, preferably, such administration is systemic(e.g., by intravenous injection). Effective amounts and effectiveperiods of time can be determined using the models and assays describedherein. For example, the examples below describe the preservation ofprogenitor cells that have SCID-reconstituting ability. In accordancewith the invention, a therapeutically effective period of time isinjecting a therapeutically effective amount of a composition and/orpharmaceutical formulation of a FRIL family member between about 5 daysbefore the patient receives treatment with a therapeutic treatment(e.g., a chemotherapeutic) having a progenitor cell-depleting activityto about 2 hours prior to treatment with the therapeutic treatment,wherein the therapeutically effective amount of a composition and/orpharmaceutical formulation of the FRIL family member is administereddaily. In accordance with the invention, a preferred therapeuticallyeffective period of time is injecting a patient (e.g., a cancer patient)with a therapeutically effective amount of a composition and/orpharmaceutical formulation of a FRIL family member between about 2 daysbefore the patient receives treatment with a therapeutic treatment(e.g., a chemotherapeutic) having a progenitor cell-depleting activityto about 1 day prior to treatment with the therapeutic treatment,wherein the therapeutically effective amount of a composition and/orpharmaceutical formulation of a FRIL is administered daily. It will beunderstood that once the patient starts to receive treatment of atherapeutic treatment having a progenitor cell-depleting activity, thetherapeutically effective amount of a composition and/or pharmaceuticalformulation of a FRIL family member may be different from thetherapeutically effective amount of the a composition and/orpharmaceutical formulation of a FRIL family member that the patientreceived prior to receiving treatment with the therapeutic treatment.

Preferably, the patient is a human or a domesticated animal.“Domesticated animal” is as described for the third aspect of theinvention.

In certain embodiments of the eighth aspect of the invention, thepatient is a cancer patient. In some embodiments, the effective amountof the composition of the FRIL family member is administered prior tothe treatment of the patient with a therapeutic treatment having ahematopoietic progenitor cell-depleting activity. In some embodiments,the therapeutic treatment is a radiotherapeutic or a chemotherapeutictreatment, including, without limitation, cytarabine (Ara-C), doxorubidn(Dox), or 5-fluorouracil (5-FU), or a combination of a radiotherapeuticand a chemotherapeutic.

In a ninth aspect, the invention provides a method for identifying aprogenitor cell, comprising contacting a candidate cell with a FRILfamily member molecule, wherein binding of the candidate cell to theFRIL family member molecule identifies the candidate cell as aprogenitor cell. “FRIL family member molecule” and “progenitor cell” areas described above for the first aspect of the invention.

In certain embodiments of the ninth aspect, the candidate cell is in apopulation of cells. In certain embodiments, the candidate cell is froma human.

In a tenth aspect, the invention provides a progenitor cell identifiedby a method comprising contacting a candidate cell with a FRIL familymember molecule, wherein binding of the candidate cell to the FRILfamily member molecule identifies the candidate cell as a progenitorcell. “FRIL family member” and “progenitor cell” are as described abovefor the first aspect of the invention.

In an eleventh aspect, the invention provides a method for identifying acomposition of a member of the FRIL family of progenitor cellpreservation factors, comprising contacting a candidate compound with aglycosylated extracellular domain of an FLT3 receptor, wherein theglycosylation pattern of the extracellular domain of the FLT3 receptoris the same as the glycosylation pattern of an extracellular domain of anormally glycosylated FLT3 receptor, wherein a candidate compound thatbinds the glycosylated extracellular domain of the FLT3 receptor isidentified as a composition of a FRIL family member. “FRIL familymember,” “progenitor cell,” and “normally glycosylated FLT3 receptor”are as described above for the first aspect of the invention.

In accordance with the eleventh aspect of the invention, the normallyglycosylated extracellular domain of the FLT3 receptor may expressed ona cell surface (e.g., on the surface of an NIH 3T3 cell, as described inthe examples below). Any normal cell may be transfected with a nucleicacid sequence encoding the extracellular domain of the FLT3 receptor(from, e.g., a human or a mouse) provided that the cell normallyglycosylates the FLT3 receptor it expresses on its cell surface. Itshould be noted that the cell need not be transfected with a nucleicacid sequence encoding the entire FLT3 receptor. For example, the cellmay be transfected with a nucleic acid molecule encoding a fusionprotein comprising the intracellular domain of a non-FLT3 receptor(e.g., the Fms receptor) and the extracellular domain of the FLT3receptor. A candidate compound according to this aspect of the inventionis then incubated with the cell expressing the normally glycosylatedextracellular domain of the FLT3 receptor, and binding of the candidatecompound to the cell (as can be measured, as described below, bysurvival of the cell) identifies the candidate compound as a FRIL familymember.

Alternatively, the normally glycosylated extracellular domain of theFLT3 receptor need not be expressed by a cell. Instead, the normallyglycosylated extracellular domain of the FLT3 receptor may be detectablylabeled or immobilized on a solid support (e.g., a magnetic bead). Forexample, the normally glycosylated extracellular domain of the FLT3receptor can be bound to the surface of a 96 well microtiter plate.Compositions comprising different candidate compounds (or pools of suchcompounds) can be detectably labeled (e.g., with biotin), and added tothe wells. After incubation for an amount of time required for bindingof a positive control (e.g., a composition of a known FRIL familymember) the plate is washed and detectably labeled avidin is added toeach well. The plate can then be read on a standard microtiter platereader to determine binding of a composition of a candidate compound tothe plate-bound normally glycosylated extracellular domain of the FLT3receptor, wherein binding (as measured by detection of the bounddetectably labeled avidin) identifies the candidate compound in thecomposition as a FRIL family member.

In certain embodiments of the eleventh aspect, the candidate compound isa lectin. In certain embodiments, the lectin is synthetic. In certainembodiments, the lectin is from a legume.

In a twelfth aspect, the invention provides an essentially purecomposition of a FRIL family member identified by the method comprisingcontacting a candidate compound with a glycosylated extracellular domainof an FLT3 receptor, wherein the glycosylation pattern of theextracellular domain of the FLT3 receptor is the same as theglycosylation pattern of an extracellular domain of a normallyglycosylated FLT3 receptor, wherein a candidate compound that binds theglycosylated extracellular domain of the FLT3 receptor is identified asan essentially pure composition of a member of the FRIL family. “FRILfamily member,” “progenitor cell,” and “normally glycosylated FLT3receptor,” and “essentially pure” are as described above for the firstaspect of the invention.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

EXAMPLE 1 Purification and Cloning of a FRIL Family Member, Dl-FRIL

Purification of Dl-FRIL from Dolichos lab lab

Seeds from the hyacinth beans (Dolichos lab lab) were purchased fromStokes Seeds (Buffalo, N.Y.) and grown in a greenhouse. Dry seeds wereground in a coffee mill and the power was extracted in 5 volumes of 50mM Tris/HCl containing 1 nM each of MgCl₂ and CaCl₂ for 4 hours at 4° C.Bean solids were pelleted by centrifugation at 10,000×g for 20 min. ThepH of the supernatant was acidified to pH 4.0 with acetic acid, followedby a second centrifugation to clarify the supernatant, and finally thepH was readjusted to 8.0 with sodium hydroxide. This crude extract wasstored at −20° C.

Single-step purification of the FRIL family member was achieved bybinding to a mannose-Sepharose matrix (Sigma). The gel (i.e., matrix)was tumbled with the thawed crude extract for 4-12 hours at 4° C.,carefully washed several times with 50 mM Tris/HCl containing 1 nM eachof MgCl₂ and CaCl₂, and then eluted with 20 mM α-methyl α-D-mannoside.Because this FRIL family member was isolated from Dolichos lab lab, itis referred to herein as Dl-FRIL.

RNA Isolation and cDNA Synthesis of Dl-FRIL

Total RNA was prepared from mid-maturation Dolichos lab lab seeds storedat −70° C. following the procedure of Pawloski et al. Mol. Plant Biol.Manual 5: 1-13,1994. Poly (A⁺) RNA was obtained from this total RNAusing the PolyATract mRNA Isolation System (Promega) according to themanufacturer's instructions. Avian myeloblastosis virus reversetranscriptase (Promega) was used to generate cDNA from 0.5 μgpoly(A⁺)RNA, or from 3.0 μg of total RNA, using 1 μg of oligo(dT) instandard reaction conditions (Sambrook et al., Molecular Cloning. ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, 1989).

Polymerase Chain Reaction and cDNA Cloning of Dl-FRIL

Based on the amino acid sequence published by Gowda et al., J. Biol.Chem. 269:18789-18793, 1994, two degenerate oligonucleotide primers weredesigned using Phaseolus codon usage (Devereux et al., Nucleic AcidsRes. 12:387-394, 1984): (SEQ ID NO:_) MLAAA(AG)TT(TC)GA(TC)CC(AT)AA(TC)CA(AG)GA(AG)GA (SEQ ID NO:_) MLZTT(AT)CC(AG)TT(TC)TGCCA(AG)TCCCA

A 500+bp product was amplified from cDNA prepared as described above, by30 cycles of polymerase chain reaction (PCR), each cycle comprising 40seconds at 94° C., 40 seconds at 50° C., 60 seconds at 72° C., followedby an extension step at 72° C. for 10 min. Reactions were performed in50 μL containing 30 pmol of each primer, 0.2 mM deoxyribonucleotides,and 0.5 unit of AmpliTaq polymerase (Perkin Elmer, Norwalk, Conn.) inthe corresponding buffer.

The 500 bp product obtained by PCR was cloned in the cloning vector,pCR2.1 (Invitrogen, Carlsbad, Calif.), and sequenced by sequenasedideoxy chain termination (United States Biochemicals) using thefollowing primers: GTACCGAGCTCGGAT (SEQ ID NO:_) TCTAGATGCATGCTCGAG.(SEQ ID NO:_)

This sequence was designated “Dl-FRILa,” as relating to the geneencoding the protein of interest, designated “Dl-FRIL” as noted above.

Based on the sequence of the Dl-FRILa amplified product, a specificprimer was prepared: MLX GTTGGACGTCAATTCCGATGTG (SEQ ID NO:_)

A degenerate primer corresponding to the first five amino acids of thesequence published by Gowda et al., J. Biol. Chem. 269:18789-18793, 1994was also prepared: MLI GC(TC)CA(AG)TC(TC)CT(TC)TC(TC)TT (SEQ ID NO:_)

The MLX and MLI primers were used in combination to amplify a 480 bpproduct from cDNA prepared as described above, through 30 PCR cyclesusing the same conditions described above. This secondary amplifiedfragment was cloned in the pCR2.1 vector and sequenced as describedabove, and was designated “Dl-FRILb.”

The 3′ end of DL-FRIL was obtained through rapid amplification of cDNAends by polymerase chain reaction (RACE-PCR) (see, e.g., Frohman “RACE:Rapid amplification of cDNA ends,” pp. 28-38 in PCR Protocols: A Guideto Methods and Applications, Innis M A, Gelfand D H, Sninsky J J, andWhite T J, eds., Academic Press, San Diego, 1990) using the 5′/3′RACEKIT (Boehringer Mannheim, Indianapolis, Ind.) according to themanufacturer's instructions. In the cDNA synthesis for the 3′ RACE, anoligo(dT) anchor primer (“AP”) supplied with the kit was used, at aconcentration of 32.5 μM, using the standard conditions describedearlier in this Example. AP GACCACGCGTATCGATGTCGAC (SEQ ID NO:_)

Nested PCR amplifications were performed using the AP anchor primer incombination with a specific primer having the following sequence: MLBAAGTTAGACAGTGCAGGAAAC. (SEQ ID NO:_)

The amplification conditions were again 30 cycles of 40 seconds at 94°C., 40 seconds at 55° C., 60 seconds at 72° C. each, with an extensionstep at 72° C. for 10 min A 900+ bp product was obtained, which wassubcloned in pCR2.1 and sequenced as described above, and was designated“Dl-FRILc” (SEQ ID NO:1).

To obtain the full length cDNA clone, the anchor primer AP was used incombination with a specific primer corresponding to the first 5 aminoacids encoded at the 5′-terminus: MLII GCACAGTCATTGTCATTTAG. (SEQ IDNO:_)

The full length cDNA was obtained through 30 cycles of PCR, each cyclecomprising 60 seconds at 94° C., 60 seconds at 58° C., 90 seconds at 72°C., with an extension step at 72° C. for 10 min. The reaction wasperformed in 100 μL containing 30 pmol of each primer, 0.2 mMdeoxyribonucleotide, 1.0 unit of Pfu polymerase (Stratagene, La Jolla,Calif.). The MLII and AP primers were designed to generate an EcoRI siteat each end (3′ and 5′) of the polynucleotide sequence. The full lengthcDNA was ligated into the EcoRI site of the cloning vector pCR2.1,resulting in the final product “pCR2.1-DLA” illustrated schematically inFIG. 1.

The Nucleotide Sequence of Dl-FRIL

The Dl-FRILc done was sequenced completely using the dideoxy chaintermination method. The nucleotide sequence of the full-length cDNA wasdetermined to be: 1 GCACAGTCAT TGTCATTTAG TTTCACCAAG TTTGATCCTAACCAAGAGGA (SEQ ID NO:1) 51 TCTTATCTTC CAAGGTCATG CCACTTCTAC AAACAATGTCTTACAAGTCA 101 CCAAGTTAGA CAGTGCAGGA AACCCTGTGA GTTCTAGTGC GGGAAGAGTG151 TTATATTCTG CACCATTGCG CCTTTGGGAA GACTCTGCGG TATTGACAAG 201CTTTGACACC ATTATCAACT TTGAAATCTC AACACCTTAC ACTTCTCGTA 251 TAGCTGATGGCTTGGCCTTC TTCATTGCAC CACCTGACTC TGTCATCAGT 301 TATCATGGTG GTTTTCTTGGACTCTTTCCC AACGCAAACA CTCTCAACAA 351 CTCTTCCACC TCTGAAAACC AAACCACCACTAAGGCTGCA TCAAGCAACG 401 TTGTTGCTGT TGAATTTGAC ACCTATCTTA ATCCCGATTATGGTGATCCA 451 AACTACATAC ACATCGGAAT TGACGTCAAC TCTATTAGAT CCAAGGTAAC501 TGCTAAGTGG GACTGGCAAA ATGGGAAAAT AGCCACTGCA CACATTAGCT 551ATAACTCTGT CTCTAAAAGA CTATCTGTTA CTAGTTATTA TGCTGGGAGT 601 AAACCTGCGACTCTCTCCTA TGATATTGAG TTACATACAG TGCTTCCTGA 651 ATGGGTCAGA GTAGGGTTATCTGCTTCAAC TGGACAAGAT AAAGAAAGAA 701 ATACCGTTCA CTCATGGTCT TTCACTTCAAGCTTGTGGAC CAATGTGGCG 751 AAGAAGGAGA ATGAAAACAA GTATATTACA AGAGGCGTTCTGTGATGATA 801 TATGTGTATC AATGATTTTC TATGTTATAA GCATGTAATG TGCGATGAGT851 CAATAATCAC AAGTACAGTG TAGTACTTGT ATGTTGTTTG TGTAAGAGTC 901AGTTTGCTTT TAATAATAAC AAGTGCAGTT AGTACTTGT

The Dl-FRIL nucleotide sequence enabled inference of the followingderived amino acid sequence for the Dl-FRIL protein: AQSLSFSFTKFDPNQEDLIF QGHATSTNNV LQVTKLDSAG NPVSSSAGRV (SEQ ID NO:2) LYSAPLRLWEDSAVLTSFDT IINFEISTPY TSRIADGLAF FIAPPDSVIS YHGGFLGLFP NANTLNNSSTSENQTTTKAA SSNVVAVEFD TYLNPDYGDP NYIHIGIDVM SIRSKVTAKW DWQNGKIATAHISYNSVSKR LSVTSYYAGS KPATLSYDIE LHTVLPEWVR VGLSASTGQD KERNTVHSWSFTSSLWTNVA KKENENKYIT RGVL

The naturally-occurring signal sequence from the FRIL family memberisolated from Dolichos lab lab (i.e., Dl-FRIL) has the followingsequence: MASSNLLTLA LFLVLLTHAN SA (SEQ ID NO: 4)

This sequence is located directly N-terminal to the first amino acid ofSEQ ID NO: 2. The nucleic add sequence of the naturally-occurringDl-FRIL protein is provided below. 1 ATGGCTTCCT CCAACTTACT CACCCTAGCCCTCTTCCTTG TGCTTCTCAC (SEQ ID NO: 3) 51 CCACGCAAAC TCAGCCGCAC AGTCATTGTCATTTAGTTTC ACCAAGTTTG 101 ATCCTAACCA AGAGGATCTT ATCTTCCAAG GTCATGCCACTTCTACAAAC 151 AATGTCTTAC AAGTCACCAA GTTAGACAGT GCAGGAAACC CTGTGAGTTC201 TAGTGCGGGA AGAGTGTTAT ATTCTGCACC ATTGCGCCTT TGGGAAGACT 251CTGCGGTATT GACAAGCTTT GACACCATTA TCAACTTTGA AATCTCAACA 301 CCTTACACTTCTCGTATAGC TGATGGCTTG GCCTTCTTCA TTGCACCACC 351 TGACTCTGTC ATCAGTTATCATGGTGGTTT TCTTGGACTC TTTCCCAACG 401 CAAACACTCT CAACAACTCT TCCACCTCTGAAAACCAAAC CACCACTAAG 451 GCTGCATCAA GCAACGTTGT TGCTGTTGAA TTTGACACCTATCTTAATCC 501 CGATTATGGT GATCCAAACT ACATACACAT CGGAATTGAC GTCAACTCTA551 TTAGATCCAA GGTAACTGCT AAGTGGGACT GGCAAAATGG GAAAATAGCC 601ACTGCACACA TTAGCTATAA CTCTGTCTCT AAAAGACTAT CTGTTACTAG 651 TTATTATGCTGGGAGTAAAC CTGCGACTCT CTCCTATGAT ATTGAGTTAC 701 ATACAGTGCT TCCTGAATGGGTCAGAGTAG GGTTATCTGC TTCAACTGGA 751 CAAGATAAAG AAAGAAATAC CGTTCACTCATGGTCTTTCA CTTCAAGCTT 801 GTGGACCAAT GTGGCGAAGA AGGAGAATGA AAACAAGTATATTACAAGAG 851 GCGTTCTGTG ATGATATATG TGTATCAATG ATTTTCTATG TTATAAGCAT901 GTAATGTGCG ATGAGTCAAT AATCACAAGT ACAGTGTAGT ACTTGTATGT 951TGTTTGTGTA AGAGTCAGTT TGCTTTTAAT AATAACAAGT GCAGTTAGTA 1001 CTTGT

A comparative illustration of the derived Dl-FRIL amino acid sequencewith the reported amino acid sequence of the mannose lectin asdetermined by Gowda et al. (J. Biol. Chem. 269:18789-18793, 1994) isshown in FIG. 2. The single sequence derived for Dl-FRIL proteincomprises domains that correspond directly and with substantial homologyto the a subunit (SEQ ID NO: ______) and β subunit (SEQ ID NO: ______)of the protein described by Gowda et al., supra. When the β subunit ofthe Gowda et al. (supra) protein is assigned to the N-terminal domainand is followed linearly by the α subunit, the arrangement of thepolypeptides shows homology to other legume lectins.

The derived Dl-FRIL amino acid sequence, however, comprises anadditional of seven amino acid residues (aa27-34) that does not occur inthe amino acid sequence described Gowda et al., supra. Several otherdifferences between the amino acid sequences of Dl-FRIL and the aminoacid sequence described by Gowda et al., supra, are also readilydiscernible from FIG. 2.

Site-Specific Mutagenesis

To establish functionality of homologs of the protein encoded by theDl-FRIL cDNA, a mutation was made in the Dl-FRIL cDNA done. The domainsof the derived protein and the pea lectin that include the mutation siteare shown below: D1-FRIL .YLNPDYG.DPNYIHIGIDV (SEQ ID NO: _) PeaFY.NAAWDPSNRDRHIGIDV (SEQ ID NO: _)

It is known that the asparagine residue (the highlighted “N”) in the pealectin is involved in binding to its saccharide ligand. Thecorresponding asparagine in Dl-FRIL (position 141 of the amino acidsequence of SEQ ID NO: 2) was mutated to aspartic acid (“D”). Thismutation was designated “N141D” for convenience.

To introduce the mutation, recombinant PCR was performed (see, e.g.,Higuchi, R., PCR Protocols: A Guide to Methods and Applications, InnisM. A., Gelfand D. H., Sninsky J. J., and White T J, eds., AcademicPress, San Diego, 1990). Two PCR reactions were carried out separatelyon the full length cDNA using two primers that contain the same mutationand produce two products with an overlapping region: MutICCATAATCGGGATCAAGATAGGTG (SEQ ID NO: _) MutII CACCTATCTTGATCCCGATTATGG(SEQ ID NO: _)

The primary PCR products were purified with the QIAquick PCRPurification kit (QIAGEN, Valencia, Calif.), according to themanufacturer's instructions. The overlapping primary products were thencombined and amplified together in a single second reaction usingflanking primers: M1 Forw AACTCAGCCGCACAGTCATTGTCA (SEQ ID NO: _)APEcoRI GAATTCGACCACGCGTATCGATGTCGAC (SEQ ID NO: _)

Both the primary and the secondary PCR reactions were performed in 100μL containing 50 pmol of each primer, 0.4 mM deoxyribonucleotide and 1.0unit Pfu polymerase (Stratagene) in the corresponding buffer. Theprimary PCR reaction amplified the two separate fragments in 30 cycles,each cycle comprising 40 seconds at 94° C., 40 seconds at 50° C., 60seconds at 72° C., with an extension step at 72° C. for 10 min. Thesecond PCR reaction amplified the recombinant fragment in 12 cyclesusing the same conditions described above.

The resulting full-length fragment contained the mutation. Therecombinant mutated product was cloned in the EcoRI site of the cloningvector pCR2.1, as illustrated schematically in FIG. 3, and sequenced asdescribed above. This plasmid is referred to as “pCR2.1-DLA(D).”

Construction of Dl-FRIL-Expressing Plant Expression Vectors andNicotiana tabacum Transformation

Recombinant PCR was used to modify the 5′ ends of both the wild-type andthe mutant Dl-FRIL clones, to introduce a signal peptide for entry ofthe protein into the endoplasmic reticulum. Following the procedure ofHiguchi, supra, the sequence encoding the signal peptide and thefull-length cDNA clones were amplified in two separate primary PCRreactions. The signal peptide sequence was obtained from theamplification of the binary vector pTA4, harboring the complete sequenceof the α-amylase inhibitor gene (Hoffman et al., Nucleic Acids Res.10:7819-7828, 1982; Moreno et al., Proc. Natl. Acad. Sci. USA86:7885-7889, 1989).

The following primers were used for amplification of the signal peptidesequence: Sigforw GAATTCATGGCTTCCTCCAAC (SEQ ID NO: _) SigrevTGACTGTGCGGCTGAGTTTGCGTGGGTG (SEQ ID NO: _)

The primers M1Forw (SEQ ID NO: ______) and AP EcoRI (SEQ ID NO: ______)used for amplification of the Dl-FRIL cDNA described above, were againused to amplify the Dl-FRIL cDNA.

The primers used for the secondary reactions were Sigforw and AP EcoRI,which were designed to generate EcoRI sites at the 5′ and the 3′ ends.Both the primary and the secondary PCR reactions were performed asdiscussed above for the site-directed mutagenesis.

The wild-type recombinant product SpDLA was cloned in the EcoRI site ofthe pbluescript SK+ cloning vector (Stratagene) to give the vectorpBS-SpDLA, as shown in FIG. 4. The mutant SpDLA(D) was cloned in thesame site of the cloning vector pCR2.1 to give the vector pCR2.1-SpM1,as shown in FIG. 5. The nucleotide sequence of each PCR product wasdetermined as described above to verify the correct attachment of thesignal peptide. The nucleotide sequence of SpDLA is defined by SEQ IDNO:22, and the derived amino acid sequence is defined by SEQ ID NO:23.

The sequences of SEQ ID NO: 22 and SEQ ID NO: 23 are as follows: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATGGCTTCCT CCAACTTACT CACCCTAGCCCTCTTCCTTG TGCTTCTCAC CCACGCAAAC   60 TCAGCCGCAC AGTCATTGTC ATTTAGTTTCACCAAGTTTG ATCCTAACCA AGAGGATCTT  120 ATCTTCCAAG GTCATGCCAC TTCTACAAACAATGTCTTAC AAGTCACCAA GTTAGACAGT  180 GCAGGAAACC CTGTGAGTTC TAGTGCGGGAAGAGTGTTAT ATTCTGCACC ATTGCGCCTT  240 TGGGAAGACT CTGCGGTATT GACAAGCTTTGACACCATTA TCAACTTTGA AATCTCAACA  300 CCTTACACTT CTCGTATAGC TGATGGCTTGGCCTTCTTCA TTGCACCACC TGACTCTGTC  360 ATCAGTTATC ATGGTGGTTT TCTTGGACTCTTTCCCAACG CAAACACTCT CAACAACTCT  420 TCCACCTCTG AAAACCAAAC CACCACTAAGGCTGCATCAA GCAACGTTGT TGCTGTTGAA  480 TTTGACACCT ATCTTAATCC CGATTATGGTGATCCAAACT ACATACACAT CGGAATTGAC  540 GTCAACTCTA TTAGATCCAA GGTAACTGCTAAGTGGGACT GGCAAAATGG GAAAATAGCC  600 ACTGCACACA TTAGCTATAA CTCTGTCTCTAAAAGACTAT CTGTTACTAG TTATTATGCT  660 GGGAGTAAAC CTGCGACTCT CTCCTATGATATTGAGTTAC ATACAGTGCT TCCTGAATGG  720 GTCAGAGTAG GGTTATCTGC TTCAACTGGACAAGATAAAG AAAGAAATAC CGTTCACTCA  780 TGGTCTTTCA CTTCAAGCTT GTGGACCAATGTGGCGAAGA AGGAGAATGA AAACAAGTAT  840 ATTACAAGAG GCGTTCTGTG ATGATATATGTGTATCAATG ATTTTCTATG TTATAAGCAT  900 GTAATGTGCG ATGAGTCAAT AATCACAAGTACAGTGTAGT ACTTGTATGT TGTTTGTGTA  960 AGAGTCAGTT TGCTTTTAAT AATAACAAGTGCAGTTAGTA CTTGT 1005 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Met AlaSer Ser Asn Leu Leu Thr Leu Ala Leu Phe Leu Val Leu Leu                5                   10                  15 Thr His AlaAsn Ser Ala Ala Gln Ser Leu Ser Phe Ser Phe Thr Lys            20                  25                  30 Phe Asp Pro AsnGln Glu Asp Leu Ile Phe Gln Gly His Ala Thr Ser        35                  40                  45 Thr Asn Asn Val LeuGln Val Thr Lys Leu Asp Ser Ala Gly Asn Pro    50                  55                  60 Val Ser Ser Ser Ala GlyArg Val Leu Tyr Ser Ala Pro Leu Arg Leu65                  70                  75                  80 Trp GluAsp Ser Ala Val Leu Thr Ser Phe Asp Thr Ile Ile Asn Phe                85                  90                  95 Glu Ile SerThr Pro Tyr Thr Ser Arg Ile Ala Asp Gly Leu Ala Phe            100                 105                 110 Phe Ile Ala ProPro Asp Ser Val Ile Ser Tyr His Gly Gly Phe Leu        115                 120                 125 Gly Leu Phe Pro AsnAla Asn Thr Leu Asn Asn Ser Ser Thr Ser Glu    130                 135                 140 Asn Gln Thr Thr Thr LysAla Ala Ser Ser Asn Val Val Ala Val Glu145                 150                 155                 160 Phe AspThr Tyr Leu Asn Pro Asp Tyr Gly Asp Pro Asn Tyr Ile His                165                 170                 175 Ile Gly IleAsp Val Asn Ser Ile Arg Ser Lys Val Thr Ala Lys Trp            180                 185                 190 Asp Trp Gln AsnGly Lys Ile Ala Thr Ala His Ile Ser Tyr Asn Ser        195                 200                 205 Val Ser Lys Arg LeuSer Val Thr Ser Tyr Tyr Ala Gly Ser Lys Pro    210                 215                 220 Ala Thr Leu Ser Tyr AspIle Glu Leu His Thr Val Leu Pro Glu Trp225                 230                 235                 240 Val ArgVal Gly Leu Ser Ala Ser Thr Gly Gln Asp Lys Glu Arg Asn                245                 250                 255 Thr Val HisSer Trp Ser Phe Thr Ser Ser Leu Trp Thr Asn Val Ala            260                 265                 270 Lys Lys Glu AsnGlu Asn Lys Tyr Ile Thr Arg Gly Val Leu        275                 280                 285A Plant Expression Vector Encoding Recombinant Dl-FRIL

A binary vector was constructed for seed-specific expression of Dl-FRIL.For seed expression, the vicilin promoter obtained from the pCW66(Higgins et al., Plant Mol. Biol. 11:683-695, 1988) was cloned inEcoRI/KpnI sites of the plant expression vector pBIN19, to formpBINVicPro, as illustrated in FIG. 6. Downstream of the vicilinpromoter, the SpDLA cDNA sequence was ligated into the EcoRI/SacI site,giving rise to the pBINVicPro-SpDLA, which is illustrated in FIG. 7. Themutated cDNA done SpDLA(D) was ligated in EcoRI site of the pBINVicProvector to yield pBINVicPro-SpDLA(D), which is illustrated in FIG. 8. Noadditional termination sequences were added, relying instead on the stopcodons and the polyadenylation site of the DLA and DLA(D) cDNA clones.Both vectors were transferred into Agrobacterium tumefaciens strainLBA4404 according to the freeze-thaw procedure reported by An et al.,“Binary vectors,” in Plant Molecular Biology Manual, Vol. A3, Gelvin SB, Schilperoort R A, and Verma D P S, eds., Kluwer Academic Publisher,Dordrecht, The Netherlands, pp. 1-19, 1988).

Agrobacterium-mediated transformation of Nicotiana tabacum leaf diskswas carried out and assayed as described (Horsch et al., Science227:1229-1231, 1985) using LBA4404 harboring the seed-specificexpression vector pBINVicPro-SpDLA (FIG. 9). Kanamycin-resistant plants(resistance being conferred by transformation with the pBIN19-basedvectors that carry the gene) were scored for their ability to form rootsin two consecutive steps of propagation in Murashige-Skoog mediumcontaining 3% of sucrose and kanamycin sulfate (Sigma, St. Louis, Mo.)at 100 mg/mL.

Recombinant Dl-FRIL fusion proteins were cleaved by the transformedplant cells in vivo. Thus, the transformed plant cells produced matureDl-FRIL which, when purified, had a molecular mass of 60 kDa andcomprised four subunits, two alpha subunits and two beta subunits (i.e.,an α₂β₂ heterodimer), where each subunit is about 15-18 kDa.

Expression of Recombinant Dl-FRIL in E. coli

The Dl-FRIL wild-type cDNA and mutant clones (without signal peptides),were ligated into the EcoRI/SalI and EcoRI/XhoI of the expression vectorpGEX 4T-1 (Pharmacia Biotech, Uppsala, Sweden), to form the expressionconstructs pGEX-M1 and pGEX M1(D), respectively illustrated in FIGS. 9and 10. The host E. coli strain, BL21(D3), was purchased from Novagen(Madison, Wis., and transformed with the above construct using thecalcium chloride method (see Sambrook et al., supra; Gelvin andSchilperoort, Plant Molecular Biology Manual, Kluwer AcademicPublishers, Dordrecht, The Netherlands, 1988; Altabella et al., PlantPhysiol 93:805-810, 1990; and Pueyo et al., Planta 196:586-596, 1995).The induction of the tac promoter (Ptac) was achieved by adding IPTG(isopropyl-β-D-thiogalactopyranoside) (Sigma) at a 1.0 mM finalconcentration when the cells reached an optical density of 0.4-0.6 at600 nm. The cultures were allowed to grow for 12 hours at 37° C. afterthe addition of IPTG. Control non-induced cultures were maintained undersimilar conditions. The cells were lysed by treatment with 4 mg/mLlysozyme in phosphate-buffered saline containing 1% TRITON® X-100.

Total cellular protein was extracted from transformed E. coli cells andanalyzed on SDS-PAGE on a 15% gel using a standard procedure (Sambrooket al., supra). The cells from 1 mL of E. coli culture were suspended inthe same volume of loading buffer (50 mM Tris HCl pH 6.8, 100 mM DTT, 2%SDS, 10% glycerol, 0.1% bromophenol blue) and vortexed. Followingtransfer to a nitrocellulose membrane, protein was stained withCoomassie Brilliant Blue R250. A representative separation is shown inFIG. 11, with the lanes identified in Table 1, below. TABLE 1 Key toFIG. 11 Lane No. Content 1 Molecular Mass Marker (Bio-Rad) 2 TotalProtein Extract from Non-Induced BL21(D3) pGEX-M1 3 Total ProteinExtract from Induced BL21(D3) pGEX-M1 4 Total Protein Extract fromNon-Induced BL21(D3) pGEX- M1(D) 5 Total Protein Extract from InducedBL21(D3) pGEX-M1(D)

The separation of proteins in FIG. 11 shows that the induced cells(lanes 3, 5) both produced an abundant polypeptide having a molecularmass of about 60 kDa (indicated by arrow). The non-induced cells failedto produce any significant amount of this protein (FIG. 11, lanes 2, 4).

Purification of Recombinant Dl-FRIL from Transformed E. coli

Induced E. coli cells (200 mL) as described above were harvested after12 hour induction at 37° C. by centrifugation at 5000 g for 10 min. Thepellet was washed with 50 mM Tris-HCl pH 8.0, 2 mM EDTA, and resuspendedin 1/10 vol of 1% TRITON surfactant in TBS (20 mM Tris pH 7.5, 500 mMNaCl). The cells were lysed by adding 4 mg/mL of lysozyme and incubatingat room temperature for 30-60 min. After centrifugation at 5000 g, thesupernatant containing the total soluble proteins was discarded and theresulting pellet, comprising the inclusion bodies and containing theaccumulated the recombinant fusion protein, was extracted with 8 Mguanidine-HCl (Martson and Hartley, “Solubilization of ProteinAggregates” in Guide to Protein Purification vol. 182, eds. Deutscher,M. P., pp 266-267, 1993).

The recombinant fusion protein solubilized by guanidine-HCl was purifiedon GST-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) accordingthe manufacturer's instructions and eluted in 1 mL of reducedglutathione (Sigma). Samples of the purified fusion proteins werecleaved with thrombin (Novagen) using 5 cleavage units/mL purifiedfusion protein.

For immunoblot analysis (Western blot), the purified proteins wereseparated by SDS-PAGE in general accordance with the method describedabove. The gel was S equilibrated in transfer buffer (25 mM Tris pH 8.3,192 mM Glycine, 20% MeOH) and blotted onto nitrocellulose (Bio-Rad,Hercules, Calif.) for 1 hour at 100 V using a Bio-Rad electrotransferapparatus. Non-specific binding was blocked by incubating the blots forat least 1 hour in 1×TBS (20 mM Tris pH 7.5,500 mM NaCl) containing 3%gelatin. Blotting was followed by incubation with a primary antibody (apolyclonal rabbit serum raised against the N-terminal peptide of theβ-subunit of Phaseolus vulgaris FRIL (i.e., Pv-FRIL), 1:100 dilution, 3hour; described below in Example 5), followed by incubation with asecondary antibody (goat anti-rabbit IgG conjugated to horseradishperoxidase at 1:1000 dilution for 1 hour). The blots were washed and thecolor developed with the color development reagent (Bio-Rad). Arepresentative result is shown in FIG. 12, with the lanes identified inTable 2, below. TABLE 2 Key to FIG. 12 Lane No. Content 1 PurifiedFusion Protein M1 2 Purified Fusion Protein M1(D) 3 Control 4 PurifiedFusion Protein M1 After Cleavage with Thrombin 5 Purified Fusion ProteinM1(D) After Cleavage with Thrombin 6 Control

The separation shown in FIG. 12 demonstrates that the two forms offusion protein have similar molecular masses of about 60 kDa, and thatthrombin cleaved both types of fusion protein to produce a newpolypeptide of molecular mass 30 kDa.

EXAMPLE 2 Recombinant Dl-FRIL Specifically Stimulates Proliferation of3T3 Cells Expressing the FLT3 Receptor

Dl-FRIL interacts with the mammalian FLK2/FLT3 tyrosine kinase receptor.A specific and quantitative biological assay using NIH 3T3 fibroblaststransfected either with a chimeric receptor having the extracellularportion of the murine FLT3 receptor combined with the intracellularportion of the human Fms receptor (Dosil et al., Mol. Cell. Biol.13(10):6572-6585 1993) or with the full length human receptor (Small etal., Proc. Natl. Acad. Sci. USA 91:459-463, 1994) can be used toevaluate lectin biological activity during purification. Serial two-folddilutions of lectin samples across rows of a 96 well plate allowed forgreater than a thousand-fold range to access FLT3 3T3 biologicalactivity. Either the murine or human FLT3 ligand (FL) (Lyman et al.,Cell 75:1157-1167, 1993; Hannum et al., Nature 368: 643-648, 1994) orthe FRIL was found to rescue FLT3-transfected cells from death in thisassay.

Specifically, 3T3 cells cultured in tissue culture plates (BectonDickinson Labware, Lincoln Park, N.J.) were removed from the plates bywashing cells twice in Hank's buffered saline solution (HBSS; GibcoLaboratories, Grand Island, N.Y.). Non-enzymatic cell dissociationbuffer (Gibco) was added for 15 minutes at room temperature. Theresulting cells wee washed in medium. FLT3 3T3 cells were cultured at afinal concentration of 3,000 cells per well in a volume of 100 μL ofserum-defined medium containing 10 mg/mL rhIL1-α, 10% AIMV (Gibco, GrandIsland, N.Y.) and 90% Dulbecco's modification of Eagle's medium (DMEM;Gibco) in 96 well plates. Under these assay conditions, cells die aftertwo to four days of culture in a humidified incubator at 37° C. and 5%CO₂ unless exogenously added ligand rescues cells from death. Each 96well plate contained wells of cells containing calf serum, whichstimulates all 3T3 cells, as a positive controls and wells of cellscontaining medium only as a negative control (“blank”). Full-lengthFms-transfected 3T3 cells (biological response shown in Tessler et al.,J. Biol. Chem., 269:12456-12461, 1994) served as receptor-transfectedcontrol target cells, and parent 3T3 cells served as untransfectedcontrol cells. Proliferation and cell survival was quantitated byaddition of XTT (2,3-bis[Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5carboxanilide inner salt) (Diagnostic Chemicals Ltd, Charlottetown,Prince Edward Island, Canada), which is a tetraformazan salt cleaved byactively respiring cells (Roehm et al., J. Immunol. Methods 142:257-265, 1991). Proliferation and cell survival was quantitatedspectrophotometrically using a Vmax kinetic plate reader (MolecularDevices Corp., Mountain View, Calif.), and recorded as either relativeactivity (units/mL) or as specific activity (units/mg). One unit ofbiological activity was defined as the reciprocal dilution at whichhalf-maximal stimulation of cells is detected.

The crude protein extract from the E. coli cultures described in Example1, above, was tested to determine whether expressed recombinant Dl-FRILpossessed any capacity to stimulate FLT3 3T3 cells using this assay. Thedata from this experiment are summarized in FIGS. 13A and 13B.Specifically, FIG. 13A is a graph showing that the crude extract of theE. coli culture containing expressed Dl-FRIL specifically stimulateshFLT3 cells; FIG. 13B is a graph showing that the same extract does notstimulate untransfected 3T3 cells. In FIGS. 13A and 13B, medium controlis represented by a solid line. The ordinate (absorbance) indicates cellviability measured by XTT at three days; the abscissa shows thereciprocal dilution of the extract sample. The apparent inhibition ofproliferation observed at higher concentrations (FIG. 13A) is notunderstood, but may relate to toxic components in the crude E. coliextract or the consequences of dose-related preservation of the 3T3fibroblasts.

EXAMPLE 3 Recombinant Dl-FRIL Preserves Mononuclear Cells andProgenitors in Liquid Culture

The recombinant Dl-FRIL protein preserved functional progenitors for atleast four weeks in liquid culture. FIGS. 14A and 14B, and Table 3illustrate the results of experiments in which recombinant Dl-FRIL wasshown to act in a dose-responsive manner to preserve human cord bloodprogenitors.

To do this, umbilical cord blood from healthy donors was collected in100 units/ml of heparin. Cord blood mononuclear cells (CB mnc) wereisolated within 4 hours of collection by FICOLL-PAQUE® separation(Pharmacia Biotech, Piscataway, N.J.) following manufacturer'sinstruction and washed in X-VIVO 10 medium (BioWhittaker, Walkersville,Md.). CB mnc were then cultured in six well tissue culture plates(Corning Inc., Corning, N.Y.) at a concentration of 200,000 cells/mL ina volume of 4 mL of X-VIVO 10 (i.e., 800,000 cells total per well).Dl-FRIL and/or recombinant E. coli Flt3-L (recFL; BioSourceInternational, Camarillo, Calif.) were added at a concentration of 40ng/ml at the outset (with no addition as a control). Cultures wereincubated in humidified chambers without medium changes for up to 29days.

After incubation, the cultured CB mnc cells were harvested by washing inX-VIVO 10 (i.e., harvested cells were pelleted and resuspended in X-VIVO10) to remove the Dl-FRIL and/or recFL, and then determining viable cellnumber by trypan blue (Sigma) exclusion. These results are shown in FIG.14A. The progenitor number and capacity of harvested cells were assessedby plating the washed cells in triplicate in fetal bovine serum-free,methylcellulose colony assay medium containing IL-2,granulocyte-macrophage CSF, and kit ligand (StemCell Technologies,Vancouver, BC, Canada). After two weeks, the resultant colonies fromeach of the triplicate wells were scored and the results are shown inFIG. 14B. Thus, FIGS. 14A and 14B show that recombinant Dl-FRILpreserved cord blood mononuclear cells and progenitors in adose-responsive manner in liquid culture.

Table 3 shows the resulting colonies after 15, 21, or 29 days ofincubation, demonstrating that Dl-FRIL, but not recFL, preservedprogenitors in suspension culture. TABLE 3 Day Medium Myeloid*Erythroid* Mix* Blast* 15 Dl-FRIL 1,033 ± 12   67 ± 12  7 ± 12 0 recFL40 ± 69 0 0 0 Dl-FRIL + 933 ± 250 167 ± 95  0 0 recFL No Addition 0 0 00 21 Dl-FRIL 387 ± 83   7 ± 12 0 167 ± 64  recFL 0 0 0 0 Dl-FRIL + 473 ±133 53 ± 42 0 300 ± 34  recFL No Addition 0 0 0 0 29 Dl-FRIL 0 0 0 80 ±72 recFL 0 0 0 0 Dl-FRIL + 0 0 0 40 ± 20 recFL No Addition 0 0 0 0*Data reported as ±SD of the three values from the triplicatemethylcellulose colony assay.The reported experiment is a representative of four experiments.

In FIGS. 14A and 14B and in Table 3, “blast” refers to coloniesconsisting of primitive, morphologically undifferentiated cells; “mix”refers to colonies consisting of myeloid and erythroid cells;“erythroid” refers to colonies consisting of erythroid cells; and“myeloid” refers to colonies consisting of myeloid cells. In FIGS. 14Aand 14B, cell number (FIG. 14A) or colony number (FIG. 14B) is shown onthe ordinate; the abscissa shows the reciprocal dilution of the sample.

As shown in Table 3, colonies derived from mature myeloid and erythroidprogenitors formed from cells cultured for 15 days in either FRIL orFRIL plus recFL; 24-fold fewer mature colonies formed from cellscultured in recFL alone; and no colonies appeared if neither waspresent. After 21 days in culture, Table 3 shows that myeloid anderythroid colonies formed only from cells exposed to Dl-FRIL. Thefrequency of myeloid colonies in Dl-FRIL-only cultures (based on theinitial number of CB mnc) decreased by 2.7 fold from 1 in 774 after 2weeks in culture to 1 in 2,067 after 3 weeks; erythroid coloniesdecreased in frequency by 9.6 fold from 1 in 11,940 to 1 in 114,287 (seeTable 3).

The colonies from this assay were photographed at various time points.As shown in FIG. 15A, in addition to myeloid and erythroid colonies, day21 cultures contained small colonies consisting of undifferentiatedcells. FIG. 15B shows that only blast-like colonies were observed whenthe cells were cultured in Dl-FRIL for 29 days (see also Table 3).

The progenitor capacity of the blast-like colonies was examined furtherfor cells initially cultured for 3 weeks in either Dl-FRIL, recFL, noaddition, or Dl-FRIL+recFL, and then without these regulators for anadditional 6 weeks in a methylcellulose colony assay. No colonies weredetected from the cells cultured for 21 days in either recFL alone ormedium control. Viable cells were harvested from the cells initiallycultured in Dl-FRIL alone and replated in an colony assay (inmethylcellulose colony assay medium) for an additional 4 weeks. Aschematic diagram of this experiment is shown in FIG. 16. Asschematically diagramed in FIG. 16, the frequency of blast-like coloniescultured in Dl-FRIL alone decreased by 2.1 fold from day 21 to day 29,from 1 in 4,790 to 1 in 10,000, and 7.5 fold in Dl-FRIL+recFL culturesfrom 1 in 2,667 to 1 in 20,000 of the initial CB mnc cells cultures.Following the protocol schematically diagrammed in FIG. 16, small,diffuse, blast-like colonies were detected at a frequency of 1 in 67(900 colonies/600,000 CB mnc) exclusively in dishes of cells initiallycultured in Dl-FRIL alone, and at a frequency of 1 in 132 (990colonies/131,000 CB mnc) for cells cultured in Dl-FRIL+recFL (seeexamplary colonies in FIG. 17).

EXAMPLE 4 Recombinant Dl-FRIL Acts Directly on Progenitor Cells

To assess whether recombinant Dl-FRIL acts directly or indirectlythrough accessory cells to preserve progenitor cells, cord bloodmononuclear cells were first enriched for progenitors expressing theCD34 antigen by immunomagnetic bead isolation (Dynal Corp., LakeSuccess, N.Y.). Five hundred CD34⁺ cells were placed into wellscontaining 100 μL of serum-free medium (BIT9500, StemCell Technologies,Vancouver, BC, Canada) either in the presence of recFL (PeproTech,Princeton, N.J.) or a cytokine cocktail of recombinant human interleukin3 (rhIL3)+recombinant human interleukin 6 (rhIL6)+recombinant humaninterleukin 11 (rhIL11)+rhTpo Thrombopoietin+FL (FLT3-Ligand) (BioSourceInternational, Camarillo, Calif.) in 96-well plates and cultured forfour weeks without medium changes. The numbers of functional progenitorsfrom these cultures were assessed by plating cells in completeserum-free methylcellulose colony assay medium (StemCell Technologies).After two weeks, the resultant colonies were scored and the results areshown in FIG. 18 (solid bars=recombinant Dl-FRIL; open bars=cytokinecocktail). Clearly, progenitors were preserved only in the recombinantDl-FRIL-containing cultures (FIG. 18). Thus, purified recombinantDl-FRIL acts directly on primitive hematopoietic progenitors.

EXAMPLE 5 Identification and Cloning of Pv-FRIL, a Second FRIL FamilyMember FRIL Activity in PHA-LCM Media

A biological screening assay to search for novel stimulators of the Flt3receptor was developed using NIH 3T3 cells transfected with expressionvectors containing cDNA of murine and human Flt3 and the related Fmsreceptor. The mFlt3/Fms 3T3 cell line is a 3T3 cell line transfectedwith nucleic acid encoding a fusion protein consisting of the murineextracellular domain of the Flt3 receptor fused to the transmembrane andintracellular domains of the human Fms (provided by Dr. Ihor Lemischka,Princeton University, Princeton, N.J.). The Stk 3T3 cell line is a 3T3cell line transfected with the full-length human Flt3 receptor (providedby Dr. Donald Small, Johns Hopkins University, Baltimore, Md.). Thehuman FMS 3T3 cell line is a 3T3 cell line transfected with thefull-length human Fms receptor (provided by Dr. Charles Sherr, SaintJude Children's Research Hospital, Memphis, Tenn. Parent 3T3 cells werepurchased from American Type Culture Collection (“ATCC”; Manassas, Va.).Receptor-transfected cells contained Neo resistance genes and weremaintained in medium containing 750 g/ml of G418 (Life Technologies,Rockville, Md.).

To create factor-dependence for receptor-transfected 3T3 cells, growthconditions were compromised to permit only ligands to rescue cells fromdeath. To do this, 3T3 cell lines were assayed in 96 well plates (BectonDickinson Labware, Lincoln Park, N.J.) containing 3,000 cells in 100 μLof serum-free medium consisting of 10% AIMV (Life Technologies) and 90%DMEM. In each experiment, samples were serially diluted two-fold acrossrows starting at a 1:10 dilution. Viable cells were quantitated after3-5 days by XTT (2,3-bis[Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5carboxanilide inner salt) (Sigma, St. Louis, Mo.) (Roehm et al., supra).Relative activity (units/ml) and specific activity (units/mg) aredefined as the reciprocal dilution at which half-maximal stimulation ofcells was detected.

The FMS 3T3 cells (transfected with cDNA encoding the human Fms tyrosinekinase receptor) and its ligand, human M-CSF, served as a model system.Recombinant human M-CSF stock of 1 μg/mL was serially diluted (where thefirst dilution, 1:20, was 50 ng/mL) was used to stimulate Fms3T3 cells.As shown in FIG. 19A, Fms 3T3 cells responded to M-CSF in adose-responsive manner. Neither the mFlt3/Fms 3T3 cells nor the parentuntransfected 3T3 cells responded to M-CSF (FIG. 19A).

Various sources of conditioned medium were screened for the presence ofFlt3 3T3 stimulatory activity. The most potent source was conditionedmedium harvested from human peripheral blood cells activated to secretehigh levels and a broad range of cytokines by the mitogenic legumelectin, phytohemagglutinin (PHA), which is derived from impure redkidney bean extracts. This source, commonly called PHAleukocyte-conditioned medium (PHA-LCM), has been used as a standardpositive control in hematopoietic colony assays for over two decades(Sharon and Lis, Science 246: 227, 1989). To make PHA-LCM, leukopherizedblood from normal volunteers was purchased from North AmericanBiologicals Inc., Miami, Fla. Mononuclear cells were isolated byFICOLL-PAQUE®, washed in AIMV, and cultured at a concentration of 2×10⁶cells/ml in AIMV containing a 1% volume of crude red kidney bean extractcontaining PHA from Life Technologies (catalog number 10576-015) ineither T150 flasks (Becton Dickinson Labware, Lincoln Park, N.J.) orroller bottles (Becton Dickinson Labware) for one week. Cells and debriswere removed by centrifugation and conditioned medium was stored at −20°C.

PHA-LCM induced proliferation of mFlt3/Fms 3T3 cells and Stk 3T3(expressing the human Flt3 receptor) in an indistinguishable manner atapproximately 200 units/mL (FIG. 19B). Untransfected 3T3 cells did notrespond to PHA-LCM (FIG. 19B) and Fms 3T3 cells responded weakly (datanot shown).

Purification of Pv-FRIL from PHA-stimulated Leukocyte Conditioned Media

Each batch of PHA-LCM was generated in serum-free medium with cells fromindividual normal donors. To start purification, the PHA-LCM with Flt33T3 activity was pooled in 30 liter lots with approximately 10⁷ Flt3 3T3units. Twenty-five liters of PHA-LCM was diafiltered into 50 mMTris-HCl, pH 7.6, 50 mM NaCl and then concentrated to 2-2.5 liters bytangential flow ultrafiltration on a 10 kDa molecular weight cutoffmembrane (Pellicon, Millipore, Bedford, Mass.). A Blue-sepharose FF(Pharmacia Biotech) column (10 cm×15 cm) was equilibrated with 50 mMTris-HCl, pH 7.4, 50 mM NaCl. To remove the human serum albumin,concentrated PHA-LCM was applied to the column at 25 ml/min by pump andthe flow through was collected. The flow-through fraction fromBlue-sepharose was subjected to anion exchange chromatography by beingapplied to a Q-sepharose FF (Pharmacia Biotech) column (5 cm×5 cm)equilibrated with 10 mM Tris-HCl, pH 7.6. The column was washed withequilibration buffer and then eluted at 12 ml/min with a continuousgradient of 0-0.7 M NaCl in 10 mM Tris. Fractions were collected (6ml/fraction) and an aliquot of each fraction was tested for Flt3 3T3activity (i.e., an ability to stimulate mFlt3/fms 3T3 cells and/or Stk3T3 cells) in 96 well plates.

After validation that a fraction had Flt3 T3 activity, aphenyl-sepharose HP (Pharmacia Biotech) column (1.6 cm×10 cm) wasequilibrated with 20 mM phosphate, pH 7, 1.5 M NH₄SO₄. The pooled samplefrom Q-sepharose was adjusted to 1.5 M NH₄SO₄ and applied to thephenyl-sepharose column. The column was washed with equilibration bufferand eluted at 1 ml/min with a gradient of 1.5-0.1 M NH₄SO₄ in 20 mMphosphate, pH 7. Fractions (1 ml) were collected and tested for Flt3 3T3activity. Fractions with Flt3 3T3 activity were pooled and dialyzedagainst 50 mM Tris-HCl, pH 7.2, 100 mM NaCl and concentrated by vacuumcentrifugation.

A Superdex 75 (Pharmacia Biotech) column (1.6 cm×60 cm) was equilibratedwith 50 mM Tris-HCl, pH 7.4, 100 mM NaCl. The pooled sample fromphenyl-sepharose was applied to the Superdex 75 column and eluted at 0.6ml/min. Fractions (1.8 ml) were collected and tested for Flt3 3T3activity. The active fractions were dialyzed against Tris-HCl, pH 7.2and concentrated by vacuum centrifugation.

A C4 reverse-phase column (4.6 mm×100 mm, Vydac, Hesperia, Calif.) wasequilibrated with 0.1% trifluoroacetic acid (TFA) in HPLC grade H₂O.Pooled and concentrated sample from Superdex 75 chromatography wasapplied to the C4 column and the column was eluted with a gradient of10-55% acetonitrile, 0.1% TFA over 70 min at 0.5 ml/min. Fractions of0.5 mL were collected and evaporated by vacuum centrifugation.

Analysis by ELISA for cytokines (IL1-α, IL1-β, IL2, IL3, IL4, IL6,GM-CSF, G-CSF and SCF) during purification of Pv-FRIL were performedusing kits purchased from R&D Systems (Minneapolis, Minn.).

Pv-FRIL Specific Rabbit Anti-Serum

Throughout purification of Pv-FRIL a New Zealand White rabbit (HRP,Denver, Pa.) was immunized with crude PHA-LCM, boosted with increasinglypurified samples containing Flt3 3T3 activity, and finally immunizedwith a peptide corresponding to Pv-FRIL (AQSLSF[N, C, S]FTKFDLD),referred to as the AQS-peptide. Samples were glutaraldehyde conjugatedto keyhole limpet hemocyanin (KLH, Sigma). The rabbit was immunized withKLH-AQS peptide-containing samples using either Complete Freund'sAdjuvant (Sigma) or Hunter's Titermax (Vaxcel, Inc., Norcross, Ga.).Antiserum demonstrated a 1:5,000 titer to the AQS peptide in an ELISA(data not shown). Since the antiserum contained reactivities to otherproteins, further enrichment for AQS peptide-specific antibodies wasachieved by either depletion of cross-reactive antibodies or by affinitypurification using a AQS peptide covalently linked to an agarose support(AminoLink coupling gel, Pierce).

An anti-AQS affinity column was prepared either by purifying IgG fromhigh titer rabbit antiserum by protein A affinity chromatography(ImmunoPure kit, Pierce) or antibody isolated from the AQS peptidecolumn and then covalently linking antibody to an activated agarosesupport (AminoLink coupling gel, Pierce).

Activity of Purified Pv-FRIL

To relate the observations of Pv-FRIL's activity on receptor-transfected3T3 cells to Flt3 receptor-expressing hematopoietic progenitors, asuspension culture of human cord blood cells enriched for Flt3⁺progenitors by CD34 immunomagnetic bead selection was adapted to a 96well plate format. To do this, umbilical cord blood from healthy donorswas collected in 100 units/ml of heparin (Fujisawa Healthcare,Deerfield, Ill.). Mononuclear cells were isolated by FICOLL-PAQUE®(Pharmacia Biotech, Piscataway, N.J.), washed in HBSS, and resuspendedin serum-defined medium, either AIMV or XVIVO-10 (BioWhittaker,Walkersville, Md.). Mononudear cells were enriched for Flt3⁺ progenitorsby CD34 immunomagnetic bead selection (Dynal Corporation, Lake Success,N.Y.). CD34⁺ cells were cultured in 6 well plates (Becton DickinsonLabware) in at a concentration of 10⁵ cells in 1 mL of DMEM containing10 ng/mL recombinant human IL3 (BioSource International, Camarillo,Calif.) and 10% fetal calf serum. The number of refractive cells presentin culture wells was scored microscopically.

Culture medium always contained IL-3 since early-acting cytokinesrequire additional co-factor(s) for survival and proliferation. FIG. 20Ashows that cord blood cells responded to column fractions in two regionsof the material eluted from an anion exchange column. The first regionof activity corresponded with Flt3 3T3 stimulatory activity (FIGS. 20Band 20C); the second associated with an activity detected with Fms 3T3cells (FIG. 20D); no response was detected in untransfected 3T3 cells(data not shown). The active material, corresponding to Flt3 3T3activity (peak one in FIG. 20A), was further characterized and purifiedon different chromatographic matrices including a cation exchange resin,heparin sepharose, hydroxyappatite, ConA-sepharose, phenyl sepharose,and gel filtration (Superdex 75) (data not shown).

The further purified Pv-FRIL was used in the Flt3 3T3 assay describedabove. Plateau stimulation of Flt3 3T3 cells decreased with sequentialpurification steps (see FIG. 21A), suggesting removal of essentialco-factor(s). Addition of suboptimal levels of crude PHA-LCM to Pv-FRILobtained in the later stages of purification, restored activity of thispartially purified Pv-FRIL to maximal plateau levels (FIG. 21B).

Analysis by ELISA for cytokines that act on hematopoietic progenitors(interleukin 1-α (IL1-α), interleukin 1-β (IL1-β), interleukin 2 (IL2),interleukin 3 (IL3), interleukin 4 (IL4), interleukin 6 (IL6),granulocyte-macrophage colony stimulating factor (GM-CSF), G-CSF(granulocyte colony stimulating factor), and stem cell factor (SCF)) infractions containing Pv-FRIL purified to near homogeneity revealed thatIL1-α had remained with Pv-FRIL through every step of purification (datanot shown).

Flt3 3T3 activity was depleted but not eliminated either by addingneutralizing antibodies to IL1 or by removing IL1 by antibody affinitychromatography (data not shown). However, at the levels (<1 ng/mL) foundin fractions containing purified Pv-FRIL, exogenous recombinant hIL1-αby itself had no stimulatory activity (data not shown). This observationsuggested the possibility that IL1-α may act as a necessary co-factor toobtain maximal stimulation by Pv-FRIL. In subsequent experiments whentesting purified Pv-FRIL, the co-factor requirement was met by theaddition of either IL1-α or PHA-LCM added at a concentration that didnot stimulate Flt3 3T3 cells by itself.

Using this modified Flt3 3T3 assay with the addition of sub-stimulatoryamounts of either IL1-α or PHA-LCM, the active protein was purified tonear homogeneity in three independent experiments. Table 4 summarizesthe results of one such experiment in which a 1% recovery wasaccompanied by an 80,000-fold purification and resulted in a fractionwith a specific activity of 244,500 units/mg. TABLE 4 Total TotalSpecific Protein Activity Activity Fold Recovery Purification Step (mg)(units) (u/mg) Purification (%) PHA-LCM 231,774 7,500,000 3 1 100 BlueSepharose FF 1,294 2,600,000 670 35 Q-Sepharose FF 347 1,400,000 4,0351,345 19 Phenyl-Sepharose HP 14 1,200,000 85,714 28,571 16 Superdex 750.12 29,340 244,500 81,500 1Pv-FRIL was purified by its ability to stimulate Flt3-expressing 3T3cells using four different chromatographic media. This resulted in a80,000-fold purification with a 1% yield.Purified Pv-FRIL

SDS-PAGE showed the purified material to contain a limited number ofpolypeptides and the molecular size of the active material wasdetermined by eluting the protein from SDS-PAGE gel slices run undernon-reducing conditions and assaying the activity of the elutedmaterial. Flt3 3T3 activity was always found in a gel slice thatcontained 14-22 kDa polypeptides and sometimes in a gel slice containing32-43 kDa polypeptides (data not shown).

The polypeptide(s) in the active fraction corresponding to the 14-22 kDarange were subjected to aminoterminal sequencing by Edman degradation.To do this, an 18 kDa species from C4 reverse-phase chromatography wasresolved by SDS-PAGE, electroblotted onto polyvinylidene difluoride(PVDF) membrane (Immobilon-P, Millipore) and stained with Ponceau S(Bio-Rad Laboratories, Inc., Hercules, Calif.). The 18 kDa band was cutfrom the PVDF membrane and N-terminal sequence was determined byautomated Edman degradation on an ABI model 477A protein sequencer (PEApplied Biosystems, Foster, Calif.). The derived peptide sequences werecompared against the SwissProt protein sequence database.

In each of three experiments, the sequence AQSLSFXFTKDALD was obtainedfrom a polypeptide of 18 kDa (where X is an unknown amino acid). For thematerial at the dye front (14 kDa and below) the aminoterminal sequenceof TDSRVVAVEFDXFP was found twice. The amino terminus of a smooth muscleprotein (SM22-α) was found twice and the amino terminus of myoglobinidentified once. Since the sequence starting with AQS was the onlysequence identified in each experiment, this polypeptide was concludedto be responsible for Flt3 3T3 activity.

Further purification of Pv-FRIL was obtained by immunoaffinitychromatography using a rabbit antiserum described above that wasgenerated against a synthetic peptide of 13 amino acids corresponding tothe N-terminus obtained for the 18 kDa polypeptide (referred to asanti-AQS). Crude PHA-LCM was applied to a rabbit anti-AQS affinitycolumn, and after washing, bound protein was eluted under acidicconditions. Four pools of fractions were assayed for activity in the twodifferent assay systems. The Flt3 3T3 cells responded weakly (<100 u/mL)to the pooled fractions (data not shown). FIGS. 22A-22D shows results ofan experiment where the pooled fractions were assayed on cord bloodcells in the presence of IL3. After two weeks of suspension culture, thenumber of viable cells and status of CD34 expression was evaluated. Arepresentative of three AQS affinity chromatography experiments is shownin FIGS. 22A-22D. Cell cultures supplemented with the two early columnfraction pools contained approximately four-fold more cells (426,000cells and 466,250 cells, respectively) than at the 100,000 cells seededand no appreciable CD34 staining (FIGS. 22A and 22B). The increase incell number and loss of CD34 expression is attributed to the expectedconsequences of IL3-induced proliferation and differentiation. Incontrast, cell cultures treated with the late-eluting fraction pool(FIG. 22D) contained less than 10,000 cells, or a tenth of input cells,and a uniform population of cells expressing CD34. The late-eluting AQSaffinity pool did not show the potent effects of IL3 (high cell numbersand exhausted medium); instead the persistence of viable cellsexpression CD34 after two weeks in suspension culture suggested that theactive component might preserve CD34⁺Flt3⁺ progenitors.

Pv-FRIL Is Derived From Phaseolus vulgaris

Because PHA is derived form red kidney bean extract and because a FRILfamily member, Dl-FRIL, was isolated from another legume, namelyDolichos lab lab, mannose-binding lectins were isolated from red kidneybean (Phaseolus vulgaris) extract using standard methods, such as theprocedure of Rudiger, H., Isolation of Plant Lectins, H.-J. Gabius andS. Gabius, eds., pp. 31-46, Berlin, 1993). The kidney beanmannose-binding lectin consisted of polypeptides with molecular weightsof 18 kDa and 15 kDa, and the aminotermini of these two polypeptidesstarted with AQSLSFXFKFDPN AND TDSRVVAVEDF, respectively (where X is anunknown amino acid).

Pv-FRIL isolated from Phaseolus vulgaris was tested for activity in theFlt3 3T3 cell assay. As shown in FIG. 23, Flt3 3T3 cells responded in adose dependent manner to Pv-FRIL, while parent untransfected 3T3 cellsdid not.

Purification of Pv-FRIL from Phaseolus vulgaris

Dry seeds from the red and white kidney beans (Phaseolus vulgaris) werepurchased from W. Atlee Burpee & Company, Warminster, Pa. Lectins wereeluted using a standard protocol. Briefly, beans were pulverized in ahome coffee grinder and added to buffer of 50 mM Tris/HCl, pH. 8.0, 1 nMeach of MgCl₂ and CaCl₂ for 4 hours at 4° C. with constant mixing. Beansolids were pelleted by centrifugation at 10,000×g for 20 min. The pH ofthe supernatant was modified to pH 4.0 with acetic acid and constantmixing to remove contaminating storage proteins, followed bycentrifugation to clarify the supernatant, and finally the pH wasreadjusted to 8.0 with sodium hydroxide before storing at −20° C.

Specific binding of Pv-FRIL to mannose enabled a single-steppurification of the lectin from the supernatant of the bean extract. 50μL extracts of either red or white kidney beans were incubated in aconical tube with 1 mL of mannose covalently bound to agarose beans(Sigma) at 4° C. with constant mixing for 4 hours to overnight. Beanswere washed gently by centrifugation (300 g, 5 min) in lectin bindingbuffer. Mannose binding protein was eluted from the mannose beans afterwashing by incubation either with 200 mM α-methyl mannoside (Sigma) or100 mM glycine, pH 2.8.

DNA Isolation and PCR amplification of Pv-FRIL-Encoding Nucleic Acid

Total genomic DNA was isolated from young Phaseolus vulgaris shootsaccording to the procedure of Dellaporta (“Plant DNA miniprep andmicroprep: Versions 2.1-2.3,”.Freeling, M. and V. Walbot (Ed.). TheMaize Handbook. XXVI+759 p. Springer-Verlag New York, Inc.: New York,N.Y., USA; Berlin, Germany.), and stored at −20° C. Based on thedetermined N-terminal amino acid sequences of Pv-FRIL, four degenerateoligonucleotides (PVbeta1, PVbeta2, PValfa1, PValfa2) were designedusing Phaseolus vulgaris codon usage. The sequences of the primers is asfollows: PVBeta1: TTY ACY AAR TTY GAY YTN GA PVBeta2: ATY TTY CAR GGWGAY GC PVAlfa1: TTR ACR TCR ATW CCR ATR TG PVAlfa2: TAR TTW GGR TCR ATRTTR GCR TT

Two sequential polymerase chain reactions (PCR) were performed. In thefirst reaction, 10 ng of bean genomic DNA was amplified by 30 cycles ofPCR, each cycle comprising 40 seconds at 94° C., 40 seconds at 50° C.,60 seconds at 72° C., and an extension step at 72° C. for 10 min. Thereactions were performed in 50 μl containing 30 pmol of each primer,PVbeta 1 and PValfa1, 0.2 mM deoxyribonucleotides and 0.5 unit ofAmpli-Taq polymerase (Perkin Elmer) in the corresponding buffer. Onemicroliter of the PCR product was amplified by 30 PCR cycles using thesame conditions as described above. The reaction was performed in 50 μLcontaining 30 pmol of the two primers, PVbeta 2 and PV alfa2, using 0.2mM deoxyribonucleotides and 0.5 unit of Ampli-Taq polymerase (PerkinElmer) in the corresponding buffer. The 460 bp fragment obtained wascloned in a T/A plasmid, pCR2.1 (Invitrogen) and sequenced by sequenasedideoxy chain termination (United States Biochemicals).

RNA Isolation and cDNA synthesis of Pv-FRIL-Encoding Nucleic Acid

Total RNA was prepared from mid-maturation Phaseolus vulgaris seedsstored at −70° C. following the procedure reported by Pawloski et al.(Mol. Plant Biol. Manual 5:1-13, 1994). The 5′/3′ RACEKIT (BoehringerMannheim) was used to generate cDNA from 5.0 μg total RNA used accordingto the manufacturer's instructions. In the cDNA synthesis for the 3′RACE, the oligo(dT) anchor primer was at the concentration of 32.5μM,.in the standard conditions. For the 5′ RACE, a specific primer(SPV1) was used at the concentration of 32.5 μM. The cDNA purificationand the subsequent tailing reaction was performed according to themanufacturer's instructions.

Polymerase Chain Reaction and cDNA cloning of Pv-FRIL-Encoding NucleicAcid

The 3′ end of Pv-FRIL was obtained through rapid amplification of cDNAends by polymerase chain reaction (RACE-PCR) using the 5′/3′ RACEKIT(Boehringer Mannheim) used according to the manufacturer's instructions.Nested PCR amplifications were performed using the PCR-Anchor primerwith the specific primers (PV3 and PV4) in two successive amplificationreactions. The sequences of these primers is as follows: PV3: CAA TGTCTT ACA ACT CAC TAA G PV4: AGT GTG GGA AGA GTG TTA TTC

The amplification conditions were 30 cycles of 40 seconds at 94° C., 40seconds at 55° C., 60 seconds at 72° C. each and an extension step at72° C. for 10 min. The reactions were performed in 50 μL containing 30pmol of each primer, PV3 and PCR-Anchor primer in the first, and PV4 andPCR-Anchor primer in the second, 0.2 mM deoxyribonucleotides and 0.5units of Ampli-Taq polymerase (Perkin Elmer) in the correspondingbuffer. The 831 bp product obtained was sub-cloned in pCR2.1 andsequenced as above reported.

For the 5′RACE, again nested PCR reactions were performed using incombination with the Anchor Primer the specific primers SPV2 and SPV3.The sequences of these primers is as follows: SPV2: ACC AAA GCT TTG GTTTTC AGA SPV3: TCT GAA AAC GTT TGA GTA GAGThe amplification conditions for both reactions were 30 cycles of 40seconds at 94° C., 40 seconds at 50° C., 60 seconds at 72° C. each andan extension step at 72° C. for 10 min. The reactions were performed in50 μL containing 30 pmol of each primer, SPV2 and PCR-Anchor primer inthe first, and SPV3 and PCR-Anchor primer in the second, 0.2 mMdeoxyribonucleotides and 0.5 unit of Ampli-Taq polymerase (Perkin Elmer)in the corresponding buffer.

To obtain the full-length cDNA done, recombinant PCR was performed(Higuchi R., supra). Two PCR reactions were carried out separately, oneon the 5′ fragment and the other on the 3′ RACE product using primerswith an overlapping region. The overlapping primary products weresubsequently re-amplified using the flanking primers resulting in afull-length fragment.

The primary PCR products were purified with the QIAquick PCRPurification kit (QIAGEN) used according to the manufacturer'sinstructions, and amplified together in a single second reaction. Forthe second PCR reaction, the primers PVEcoRI and the APEcoRI were used.The sequences of these primers is as follows: PVEcoRI TAC ATG AAT TCGCTC AGT CAT TAT CTT TTA AC APEcoRI: GAA TTC GAC CAC GCG TAT CGA TGT CGAC

Both primary and secondary PCR reactions were performed in 100 μLcontaining 50 pmol of each primer, 0.4 mM deoxyribonucleotide and 1.0unit Pfu polymerase (Stratagene) in the corresponding buffer. Theprimary PCR reaction amplified the two separate fragments by 30 cycles,each cycle comprising 40 seconds at 94° C., 40 seconds at 50° C., 60seconds at 72° C. and an extension step at 72° C. for 10 min. The secondPCR reaction amplified the recombinant fragment in 12 cycles using thesame conditions reported above. The full lengthed product was cloned inthe EcoRI site of the cloning vector pCR2.1 (FIG. 24A) and sequenced asnoted above. This plasmid is referred to as pCR2.1-Pv-FRIL.

The nucleic acid sequence of the Pv-FRIL cDNA is as follows: 1GCTCAGTCAT TATCTTTTAA CTTTACCAAG (SEQ ID NO: 5) TTTGATCTTG ACCAAAAAGA 51TCTTATCTTC CAAGGTGATG CCACTTCTAC AAACAATGTC TTACAACTCA 101 CTAAGTTAGACAGTGGAGGA AACCCTGTGG GTGCTAGTGT GGGAAGAGTG 151 TTATTCTCTG CACCATTTCATCTTTGGGAA AACTCTATGG CAGTGTCAAG 201 CTTTGAAACT AATCTCACCA TTCAAATCTCAACACCTCAC CCTTATTATG 251 CAGCTGATGG CTTTGCCTTC TTCCTTGCAC CACATGACACTGTCATCCCT 301 CCAAATTCTT GGGGCAAATT CCTTGGACTC TACTCAAACG TTTTCAGAAA351 CTCCCCCACC TCTGAAAACC AAAGCTTTGG TGATGTCAAT ACTGACTCAA 401GAGTTGTTGC TGTCGAATTT GACACCTTCC CTAATGCCAA TATTGATCCA 451 AATTACAGACACATTGGAAT CGATGTGAAC TCTATTAAGT CCAAGGAAAC 501 TGCTAGGTGG GAGTGGCAAAATGGGAAAAC GGCCACTGCA CGCATCAGCT 551 ATAACTCTGC CTCTAAAAAA TCAACTGTTACTACGTTTTA TCCTGGGATG 601 GAAGTTGTGG CTCTCTCCCA TGATGTTGAC TTACATGCAGAGCTTCCTGA 651 ATGGGTTAGA GTAGGGTTAT CTGCTTCAAC TGGAGAGGAG AAACAAAAAA701 ATACCATTAT CTCATGGTCT TTCACTTCAA GCTTGAAGAA CAACGAGGTG 751AAGGAGCCGA AAGAAGACAT GTATATTGCA AACGTTGTGC GATCATATAC 801 ATGGATCAATGACGTTCTAT CTTATATAAG CAATAAATAA ATGTATGATG 851 CACTCAATAA TAATCACAAGTACGTACGGT GTAGTACTTG TATGTTGTTT 901 ATGAAAAAAA AAAA

The amino acid sequence of Pv-FRIL is as follows:AQSLSFNFTKFDLDQKDLIFQGDATSTNNVLQLTKLDSGGNPVGASVGRVLFSAPFHLWENSMAV (SEQID NO: 6)SSFETNLTIQISTPHPYYAADGFAFFLAPHDTVIPPNSWGKFLGLYSNVFRNSPTSENQSFGDVNTDSRVVAVEFDTFPNANIDPNYRHIGIDVNSIKSKETARWEWQNGKTATARISYNSASKKSTVTTFYPGMEVVALSHDVDLHAELPEWVRVGLSASTGEEKQKNTIISWSFTSSLKNNEVKEPKEDMYIANVVRSYTWINDVLSYISNK*MYDALNNNHKYVRCSTCMLFMKKK

The amino acid sequence of Pv-FRIL was compared to the amino acidsequences of Dl-FRIL and of the PHA-E lectin. This comparison is shownon FIG. 24B.

Pv-FRIL-Encoding Plant Expression Vectors and Nicotiana tabacumTransformation

Recombinant PCR was used to introduce a signal peptide for entry ofPv-FRIL into the endoplasmic reticulum at the 5′ end of the Pv-FRIL cDNAclone. Following the procedure of Higuchi (supra) the signal peptide andthe full length cDNA clone were amplified in two separate primary PCRreactions. The signal peptide was obtained from the amplification of thebinary vector pTA4, harboring the complete sequence of the beanα-amylase inhibitor gene (Hoffman et al., L. M., Y. Ma and R. F. Barker,Nucleic Acid Res. 10: 7819-7828, 1982; Moreno and Chrispeels, Proc.Natl. Acad. Sci. USA 86: 7885-7889, 1989).

The primers used for the two primary reactions are the following:Amplification of the Signal Peptide Sigforw AGA TCT ATG GCT TCC TCC AACBglII: Sigrew: AAA GAT AAT GAC TGA GCG GCT GAG TTT GCG TG

Amplification of the mannose lectin cDNA: SpMlforw: CAC GCA AAC TCA GCCGCT CAG TCA TTA TCT TT APXhoI: CTC GAG GAC CAC GCG TAT CGA TGT CGA

The primers used for the secondary reactions, Sigforw and AP, weredesigned to generate BglII sites at the 5′ and XhoI at the 3′ ends.Both, primary and secondary PCR reactions were performed as discussedabove. The recombinant product SpPv-FRIL was incubated for 10 min. at72° C. with 0.5 units of Ampli-Taq polymerase (Perkin Elmer) and clonedin the cloning vector pCR2.1 (FIG. 25). The nucleotide sequence of thePCR product was determined as described above to verify the correctattachment of the signal peptide.

A binary vector was constructed for constitutive expression of Pv-FRILin tobacco plants. The recombinant SpPv-FRIL was cloned in BglII/XhoIsites of a plant expression vector resulting in the formation ofpM-SpPv-FRIL (FIG. 26).

The binary vector was transferred in Agrobacterium tumefaciens strainC58 according to the freeze-thaw procedure reported of An et al., supra.Agrobacterium-mediated transformation of Nicotiana tabacum leaf diskswas carried out as described by Horsh et al. (Science 227: 1229-1231,1985) using C58 harboring the expression vector pM-SpPv-FRIL. Kanamycinresistant plants were scored for their ability to form roots in twoconsecutive steps of propagation in Murashige-Skoog medium containing 3%sucrose and kanamycin sulfate (Sigma) at 100 mg/L.

Tobacco plants transformed with this construct were grown in a growthroom under controlled conditions. The leaves (20 grams) of young plantswere harvested and frozen in liquid nitrogen and powdered in a mortarwith a pestle. The powder was stirred in a buffer mixture consisting of1×phosphate buffered saline containing 1 mM CaCl₂ with a cocktail ofprotease inhibitors (PMSF, Pepstatin and Leupeptin). This slurry wascentrifuged at 2000 rpm and the supernatant was centrifuged at 40,000rpm in a Beckman ultracentrifuge. The clear supernatant was tumbled with1 ml of ovalbumin-Sepharose for 3 hours. The beads were washed with thesame buffer and tumbled overnight with 200 mM trehalose in 1/10phosphate buffered saline containing the cocktail of proteaseinhibitors. Coomassie blue stained gel showed this preparation to bepure Pv-FRIL. An immunoblot showed the presence of both alpha and betasubunits, thus, the single polypeptide chain encoding both subunits wascleaved by the transformed cells in vivo. Binding to theovalbumin-sepharose and release by trehalose shows that the product ofthe transgene is an active lectin.

EXAMPLE 6 Dl-FRIL Supports Prolonged ex vivo Maintenance of OuiescentHuman CD34+,CD38−/SCID-Repopulating Cells

To further characterize the progenitor cell preservation activity ofDl-FRIL, a functional in vivo assay for primitive human hematopoieticcells was used to determine the cells' ability to home to and repopulatethe marrow of sublethally irradiated C.B-17 and NOD/LtSz mice homozygousfor the severe combined immunodeficiency Prkdc^(scid) mutation (Lapidotet al., Science 255:1137, 1992; Larochelle et al., Nat. Med.2:1329-1337, 1996). To do this, the following methods were used:

Preparation of Human Cells

Human umbilical cord blood (CB) samples were obtained from full termdeliveries. The blood samples were diluted 1:1 inphosphate-buffered-saline (PBS) without Mg⁺²/Ca⁺², supplemented with 10%fetal bovine serum (FBS). Low density mononuclear cells were collectedafter standard separation on Ficoll-Paque (Pharmacia Biotech, Uppsala,Sweden), and washed in RPMI with 10% FBS. Some samples were frozen in10% DMSO, while the others were used fresh. Enrichment of CD34⁺ cellswas performed with mini MACS separation kit (Miltenyi Biotec, BergischGladbach, Germany) according to the manufacturer's instructions. Thepurity of the enriched CD34⁺ cells was 60-80% using one column.CD34⁺CD38^(−/low) cells were purified by FACS sorting (FACStar⁺, BectonDickinson, San Jose, Calif.) after staining CD34⁺ enriched cells withmAb anti human CD34-FITC (Becton Dickinson) and anti human CD38 PE(Coulter, Miami Fla. USA) (Purity>99%).

Mice

Eight week old NOD/LtSz-Prkdc^(scid)/Prkdc^(scid) (NOD-SCID) mice andNOD/SCID β2 microglobulin knockout mice, hereafter termed NOD/SCIDB2M^(null) (Christianson et al., J. Immunol. 158:3578-3586, 1997), bredand maintained under defined flora conditions in sterile micro isolatorcages, were irradiated with a sublethal dose of 375 cGy at 67 cGy/min.from a cobalt (⁶⁰Co) source prior to transplantation.

Human cells were injected into the tail vein of irradiated mice in 0.5mL of RPMI with 10% FBS. In some experiments (as indicated) nonengrafting irradiated (1500 cGy) CD34⁻ cells served as carrier cells,and were cotransplanted with cultured cells at a final concentration of0.5×10⁶ cells/mouse. Mice were sacrificed 1 month post transplantation,and bone marrow (BM) cells were flushed from the 8 bones of each mouse(femurs, tibias, humeri, and pelvis).

Ex vivo Cultures

Human CD34⁺ enriched cells were cultured in 24 well plates (2-4×10⁵cells in 0.5 mL), containing RPMI supplemented with 10% FBS+1% BSA. Exvivo cultures contained the following cytokine combination: Stem cellfactor (SCF)-100 ng/mL and Flt3 ligand (Flt3-L)-100 ng/mL (R&D SystemsInc. Minneapolis, Minn., USA), rhIL-6-50 ng/mL and sIL6R-1280 ng/mL,(InterPharm Laboratories, Ares-Serono Group, Ness Ziona, Israel) orDl-FRIL-10 ng/mL (ImClone Systems Inc., NY, USA). In some experimentswhere indicated, the growth factor cocktail included 300 ng/mL SCF, 300ng/mL Flt3-L, 50 ng/mL G-CSF, 10 ng/mL IL-3 (R&D Systems), and 10 ng/mLIL-6. The cultures were incubated at 37° C. in a humidified atmospherecontaining 5% CO₂. After 3, 6, 10 and 13 days, the cells were,collected, counted, analyzed by FACS, seeded in to semisolid culturesand transplanted into NOD-SCID mice. For CD56⁺ NK cell development, BMcells from engrafted mice were cultured with 100 ng/mL of SCF and IL-15(R&D) for 10-14 days.

Preparation of Dl-FRIL

Dl-FRIL was isolated from the seeds of hyacinth beans (Dolichos lab lab)using the protocol described above in Example 1.

CFU Assay.

Semisolid cultures were performed in order to detect the levels of humanprogenitors in ex vivo cultures, and in the marrow of transplanted mice.The cells were plated (4×10³ cells/mL) in 0.9% methylcellulose (Sigma,St. Louis, Mo., USA), 30% FBS, 5×10⁻⁵M 2ME, 50 ng/mL SCF, 5 ng/mL IL-3,5 ng/mL GM-CSF (R&D), and 2 u/mL Erythropoietin (Ortho Bio Tech, DonMills, ON, Canada). Human cells from the BM of engrafted mice wereplated (2×10⁵ cells/mL) in 15% FBS+15% human plasma, selective for humancolonies only. The cultures were incubated at 37° C. in a humidifiedatmosphere containing 5% CO₂ and scored 14 days later with an invertedmicroscope for myeloid, erythroid, and mixed colonies by morphologiccriteria.

Cell Cycle Analysis.

Cells were analyzed for their DNA content by staining with propidiumiodide (Sigma). The cells were cultured in ex vivo cultures asdescribed, for 3, 6, 10, and 13 days as indicated. At each time point,the cells were collected, resuspended to a final concentration of0.1-1×10⁶ cells/mL, and incubated with 0.1% Triton×100 (Sigma) for 20minutes on ice. 50 mg/mL propidium iodide (PI) were added beforeanalysis. Flow cytometeric analyses were performed using FACSort (BectonDickinson, San Jose, Calif.).

Flow Cytometry Analyses.

Human and mouse Fc receptors on BM cells from transplanted mice wereblocked by using human plasma (1:50) and anti mouse Fc receptor blockers(anti mouse CD16/CD32 mAb, Pharmingen, San Diego, Calif., USA). Isotypecontrol mAb were used in order to exclude false positive cells (Coulter,Miami Fla. USA). The purity of enriched subpopulations after magneticbead separation, were analyzed by two color staining, using anti-humanCD34 FITC (Becton Dickinson, San Jose, Calif., USA) and anti-human CD38PE (Coulter). The levels of human cells and lymphoid lineages in themarrow of engrafted mice were detected by double staining withanti-human CD45 FITC (Immuno Quality Products, Groningen, TheNetherlands) together with anti-human CD19 PE (Coulter) for detection ofpre B cells, or with anti-human CD56 PE (Coulter) for detection of NKcells. Cells were washed with PBS supplemented with 1% FBS and 0.02%azide, suspended to a volume of 0.1-1×10⁶ cells/mL, stained withdirect-labeled mAb and incubated for 25 minutes on ice. After staining,cells were washed once in the same buffer and analyzed on a FACSort(Becton Dickinson). Analysis was performed using CELLquest software(Becton Dickinson).

Human Cell Engraftment Analysis.

The levels of human cell engraftment were determined by both flowcytometry for analysis of human myeloid CD45⁺ and lymphoid CD45⁺CD19⁺pre B cells and quantification of human DNA as previously described(Lapidot et al., Science 255:1137, 1992; Larochelle et al., Nat. Med.2:1329-1337, 1996; Peled et al., Science 283:845-848, 1999). Briefly,high molecular weight DNA was obtained from the BM of transplanted miceby phenol/chloroform extraction. DNA (5 g) was digested with EcoRI,subjected to electrophoresis on 0.6% agarose gel, blotted onto a nylonmembrane, and hybridized with a human chromosome 17-specific α-satelliteprobe (p17H8) labeled with ³²P (Lapidot et al., supra). The intensity ofthe bands in the samples were compared to artificial human/mouse DNAmixtures (0%, 0.1%, 1%, and 10% human DNA) to quantify the human DNA(lanes to the right of lane 4 in FIG. 31). Multiple exposures of theautoradiographs were taken to ensure sensitivity down to 0.01% humanDNA. A transplanted mouse was scored positive when both human myeloidand lymphoid cells and human DNA were detected in its BM.

From these experiments, the following results were obtained:

Dl-FRIL Maintains but does not Expand CB CD34⁺ Progenitors in SuspensionCulture

As shown above, Dl-FRIL by itself preserves immature CB progenitor cellsup to a month in suspension culture without medium changes (see, e.g.,FIGS. 14A and 14B, and Table 3). To compare Dl-FRIL'sprogenitor-preserving properties to a combination of cytokines (SCF,Flt3-L, IL-6, and sIL6-R) shown to maintain CB progenitor cells insuspension culture (Sui et al., Proc. Natl. Acad. Sci. USA 92:2859,1995; Ebihara et al., Blood 90:4363-4368, 1997), enriched CB CD34⁺ cellswere cultured with either Dl-FRIL or cytokines for 3,6, 10, or 13 daysin medium containing 10% FBS and 1% BSA in RPMI. Fresh media and Dl-FRILand/or cytokines were added on day 6. Cells harvested at each time pointwere counted and assayed for clonogenic progenitor cells by plating(i.e., seeding) in semisolid media.

As shown in FIG. 27A, the total number of cells in Dl-FRIL culturesgradually declined over time from 2×10⁵ cells initially seeded to1.26×10⁵ cells at day 13, in contrast to the expected 14.3-fold increaseto 2.8×10⁶ cells in cytokine cultures at day 13. Similarly, as shown inFIG. 27B, the levels of progenitor cells in Dl-FRIL cultures alsoremained relatively constant until day 10, after which they declined to9% of the starting population (from 24.7×10³ colonies on day 0 to2.3×10³ colonies on day 13). Progenitor levels increased in cytokinecultures by 10-fold from the outset to day 13 of culture (FIG. 27B).

Dl-FRIL Maintains the Expansion Capacity of CD34⁺ Progenitors up to 2Weeks in ex vivo Culture

To characterize the expansion capacity of Dl-FRIL-preserved progenitorcells in suspension culture, CD34⁺ cells cultured for 6 days withDl-FRIL were washed and exposed to cytokines (without Dl-FRIL) for anadditional 4 days. Total cell counts and clonogenic progenitor assayswere performed and the results were compared to cells cultured for 10days with Dl-FRIL. Fresh media and Dl-FRIL were added on day 6.Interestingly, the Dl-FRIL-cultured cells proliferated in response tocytokine stimulation, resulting in a 3.4-fold increase in cell numbers(FIG. 28A) and 13-fold increase in progenitor levels (FIG. 28B).

Further experiments tested Dl-FRIL's ability to preservecytokine-responsive CD34⁺ progenitor cells cultured with either Dl-FRILfor 10 days followed by 3 days of cytokine stimulation or only withDl-FRIL for the entire 13 days. Fresh media and Dl-FRIL were added onday 6. A 2.9-fold increase in total cell numbers (FIG. 28C) and a5.5-fold increase in progenitor levels (FIG. 28D) were observed forcells cultured with Dl-FRIL for 10 days followed by an additional 3 daysof cytokine stimulation compared to cultures with Dl-FRIL alone for 13days. These results provide evidence that Dl-FRIL alone maintains theproliferative capacity of human progenitor cells up to 13 days insuspension culture and that subsequent stimulation of cells cultured for10 days with Dl-FRIL still respond to the proliferative signals of thecytokines.

Dl-FRIL Maintains SCID Repopulating Stem Cells (SRC) in ex vivo Cultures

At each time point during culture, the human CB CD34⁺ cells werecollected and the content of each well was assayed for progenitor levels(described above) and 2×10⁵ of the remaining cells were transplantedinto one mouse. One month later, mice were sacrificed and their bonemarrows were harvested and assayed for the presence of human myeloid andlymphoid cells. Before assaying, human DNA levels in the bone marrow ofindividual transplanted mice was quantitated by Southern blottinganalysis. A representative Southern blot analysis showing the detectionof a 0%, 0.1%, 1%, and 10% human DNA per murine DNA is provided in FIG.29E.

FIGS. 29A-29D show representative Southern blot analyses of the BM ofmice transplanted with ex vivo cultured cells. FIG. 29A is arepresentative Southern blot showing human DNA in the marrow of micetransplanted with cells cultured with FRIL for 6 days (lane 1), FRIL for10 days (lane 2), or with FRIL for 6 days followed by 4 days withcytokine stimulation (lane 3). In a typical experiment, the levels ofengraftment by cells cultured with Dl-FRIL for 10 days decreased byapproximately 10-fold from cells cultured with Dl-FRIL for 6 days (FIG.29A, lanes 1 and 2). However, when cells cultured with Dl-FRIL for 6days were subsequently stimulated with cytokines for 4 days, there wasan approximate 10-fold increase in the level of engraftment (FIG. 29A,lane 3) compared to Dl-FRIL alone for 10 days (lane 2). Similar resultswere obtained when cells from other donors were transplanted as shown inFIG. 29B, lanes 3-4 (cells cultured with Dl-FRIL for 6 days followed by4 days of cytokine stimulation) compared to lanes 1-2 (cells culturedwith Dl-FRIL alone for 10 days) prior to transplantation. Dl-FRILcultures could be prolonged even to 13 days, as shown in FIGS. 28C-28D,prior to transplantation into mice. A modest increase in the levels ofengraftment was seen comparing the marrow of mice transplanted with theoriginal cells, which were not cultured with Dl-FRIL, prior to seeding(FIG. 29C, lane 1) with cells cultured with Dl-FRIL for 13 days (FIG.29C, lane 2). FIG. 28D shows the results of another experiment, wherethe difference between engraftment levels obtained by cells culturedwith Dl-FRIL for 10 days (lane 1) or with Dl-FRIL for 6 days followed by4 days of cytokine stimulation (lane 2) are minor. Nevertheless, a10-fold increase was observed for cells cultured either with Dl-FRIL for13 days (FIG. 29D, lane 3) or with Dl-FRIL for 10 days followed by 3days of cytokine stimulation (FIG. 29D, lane 4) when compared to cellscultured for 10 days (FIG. 29D, lanes 1 and 2).

Levels of human engraftment in the marrow of mice transplanted withCD34⁺ cells cultured with either Dl-FRIL for 10 days (n=12) or Dl-FRILfor 6 days (n=33) followed by cytokine stimulation for 4 days aresummarized in FIG. 30. Cells cultured with Dl-FRIL followed bycytokines, engrafted at levels 7.4-fold greater than cells cultured withDl-FRIL alone for 10 days (FIG. 30, p=0.05).

To test the effect of Dl-FRIL on CB populations highly enriched forprimitive human SRC, CD34⁺ cells isolated by immunomagnetic beads werefurther sorted by flow cytometry based on the absence of CD38expression. CD34⁺CD38^(−/low) cells (99% purity) cultured with eitherDl-FRIL alone or Dl-FRIL followed by cytokines showed patterns ofengraftment in NOD/SCID B2M^(null) mice (Christianson et al., supra),similar to those observed for CD34⁺ cells. These mice have been usedsuccessfully for secondary human stem cell transplantation (Peled etal., supra), have less innate immunity due to lack of NK activity andthus require fewer (10 fold) human cells for engraftment compared toNOD/SCID mice.

FIG. 31 is a representative Southern blot (1 out of 4 experiments)showing the relative level of engraftment of cells that were culturedwith Dl-FRIL for 10 days (lane 4) compared to 6 days with Dl-FRILfollowed by 4 days cytokine stimulation (lanes 1-3) prior totransplantation into mice. High molecular weight DNA was obtained fromthe BM of transplanted mice and subjected to Southern blotting analysisfor the presence of human DNA.

Based on these results, an increased level of engraftment in cellsexposed to cytokines after Dl-FRIL was expected. Consequently, the sameinitial cell dose transplanted into one mouse in lane 4 was divided into3 mice in lanes 1-3, indicating an expansion of Dl-FRIL cultured SRCwhen they were subsequently exposed to cytokine stimulation. The levelof engraftment in mice transplanted with cells that were treated withDl-FRIL followed by cytokines (FIG. 31, lanes 1-3) was about 10-foldhigher compared to the mouse transplanted with cells cultured withDl-FRIL alone (FIG. 31, lane 4). Since the initial dose of cells for theDl-FRIL +cytokines sample was split into three mice, a 30-fold increasein the level of engraftment is observed for CD34⁺CD38^(−/low) cellsstimulated by cytokines after Dl-FRIL compared to the culture withDl-FRIL alone. In addition, this result represents about a 3-foldgreater level of expansion of SRC for CD34⁺CD38^(−/low) cells than fortotal CD34⁺ cells. FIG. 32 summarizes the fold increase observed in theengraftment levels of CD34 ⁺CD38^(−/low) cells cultured with Dl-FRILalone compared to cells subsequently stimulated with cytokines in 6experiments (3 experiments with NOD/SCID B2M^(null) mice and 3experiments with NOD/SCID mice, gave similar results). These dataindicated a 30-fold expansion of SRC for CD34⁺CD38^(−/low) cells and a3-fold greater level than that of total CD34⁺ cells in the correspondingexperiments.

Further studies have demonstrated that Dl-FRIL preserves long-termrepopulating stem cells. Bone marrows harvested from NOD/SCID mice thatwere initially transplanted with CD34+ cord blood cells cultured inDl-FRIL were transplanted into a second set of sublethaly irradiatedNOD/SCID mouse recipients. After one month, bone marrows from the secondset of recipients were analyzed for the presence of human hematopoiesis,as determined using the assays described above. Multilineage engraftmentwas observed in the second set sublethaly irradiated NOD/SCID mouserecipients. This observed persistence of repopulating cells in thisserial transplantation study indicated the presence of long-termrepopulating stem cells.

Dl-FRIL Preserves SRC Potential of Multilineage Differentiation in theMurine BM

Southern blot analysis detected and quantified the levels of human DNAin the marrow of transplanted mice without specifically indicating thedifferentiation status of human cells recovered from the murine BM.Since SCID repopulating stem cells (SRC) are defined by multilineagedifferentiation of myeloid and lymphoid cells in NOD/SCID mice, the invivo differentiation processes of Dl-FRIL-cultured CB CD34⁺ cells in themurine BM were further studied. To do this, CD34⁺ or sortedCD34⁺CD38^(−/low) cells were cultured either with FRIL for 10 days orwith FRIL for 6 days followed by cytokine stimulation for 4 days priorto transplantation. BM of transplanted mice was collected 1 month laterand the cells were either seeded in semisolid media selective for humancolonies (results shown in FIG. 33A) or were analyzed for lineagespecific markers by flow cytometry (results shown in FIGS. 33B-33F).

The progenitor capacity of human CB CD34⁺ cells cultured for 10 dayswith Dl-FRIL and harvested one month after transplantation into NOD/SCIDmice was evaluated in semi solid-colony assays that selectively promotedgrowth of human progenitors. FIG. 33A shows that the levels of humanprogenitors in the bone marrow of transplanted mice increased 2.3-foldwhen SRC were stimulated with cytokines for 4 days after 6 days ofDl-FRIL incubation compared to Dl-FRIL alone for 10 days prior totransplantation. Moreover, both myeloid and erythroid colonies formed inthe colony assays (data not shown) as well as human B celldifferentiation that was determined by flow cytometry (FIGS. 33C and33D), indicating that incubation with Dl-FRIL maintained themultilineage differentiation potential of SRC. A representative flowcytometry analysis demonstrated the presence of human CD45⁺CD19⁺ pre-Bcells in the marrow of mice transplanted with either CD34⁺ cells (FIG.33C) or CD34⁺CD38^(−/low) cells (FIG. 33D) cultured with Dl-FRIL for 10days (1% or 10% CD19⁺ cells, respectively).

To determine whether human lymphoid precursors in the marrow oftransplanted mice have the potential to differentiate in addition tomyeloid cells also into lymphoid NK cells, cells from transplantedmurine bone marrow were also cultured with SCF and IL-15 for 10 days.Cells harvested from cultures were analyzed for human NK cells by flowcytometry using the human-specific monoclonal antibodies to CD45 and theNK cell antigen, CD56. Cells from mice transplanted with CD34 ⁺ orCD34⁺CD38^(−/low) cells are shown (1% or 7.7% of CD56⁺ cells; FIGS. 33Eand 33F, respectively).

ex vivo Preservation of Early Progenitors with Dl-FRIL Compared to Flt3Ligand

Dl-FRIL was identified by its ability to stimulate proliferation of NIH3T3 cells transfected with Flt3 and not by untransfected cells or cellstransfected with the related Fms tyrosine kinase receptor (Moore et al.,Blood 90 supp. 1:308, 1997). Although Dl-FRIL and Flt3-L both stimulateproliferation of Flt3 3T3 cells, they exert different activities on CBprogenitor cells. Flt3-L induces quiescent primitive cells into cyde(Lyman and Jacobsen, Blood 91:1101-11345, 1998). In contrast, Dl-FRIL byitself maintains progenitors in serum-defined medium from 15-29 days inculture without medium changes (Colucci et al., Proc. Natl. Acad. Sci.USA 96(2):646-50, 1999). The abilities of Dl-FRIL or Flt3-L to maintainCB CD34 ⁺ progenitors in suspension cultures consisting of 10% FBS and1% BSA in RPMI for 10 days were compared in the presence and absence ofcytokines. The cells were then were seeded for CFU assay.

As shown in FIG. 34A, the number of total cells harvested after 10 daysof suspension culture in Dl-FRIL increased minimally by 1.9-fold,whereas cells cultured in either Flt3-L alone or in combination withDl-FRIL increased by 4.5-fold and 5.4-fold, respectively (p<0.05).Cultures containing CB CD34⁺ cells with cytokines, either alone or withDl-FRIL and Flt3-L, led to 10.9-12.5 fold increase in cell numbers;however cells cultured with Dl-FRIL alone followed by cytokines,increased only by 4.6-fold (FIG. 34A, p<0.05).

Interestingly, cells cultured with Dl-FRIL alone or followed bycytokines, selectively maintained a higher number and proportion of theprimitive granulocyte-erythroid-macrophage-megakaryocyte colonyforming-units (CFU-GEMM) compared to other treatments (FIG. 34B,p<0.05). About 40% of the progenitor cells maintained by Dl-FRIL wereCFU-GEMM, a relative 2.6- to 3.7-fold increase with Dl-FRIL culturescompared to other combinations (FIG. 34B, p<0.05). In contrast toFlt3-L, exposure of cells first cultured with Dl-FRIL to cytokines,maintained the high percentage of CFU-GEMM, 18.2% versus 38.6%,respectively (FIG. 34B p<0.05). These results demonstrate that Dl-FRILand Flt3-L may act differently on primitive progenitor cells in culture.

Dl-FRIL Maintains Higher Levels of CD34⁺ Cells in G₀/G₁ Phase of CellCycle Compared to Cytokine Treated Cells

In vivo, immature CD34⁺ cells are predominantly quiescent, non-cyclingcells (Young et al., Blood 87:545, 1996; Movassagh et al., Stem Cells15:214-222, 1997; Ladd et al., Blood 90:658-668, 1997). Since Dl-FRILmaintains relatively constant levels of progenitor cells over two weeksin suspension culture that can subsequently respond to cytokinestimulation (see FIG. 27), the cell cycle status of CD34⁺ cellsincubated with Dl-FRIL was investigated and compared to cultures withcytokine stimulation. DNA content of CB CD34⁺ cells was analyzed by flowcytometry immediately after isolation of cells. A mean value of 96.6% ofcells were observed in the G₀/G₁ phase of cell cycle (FIG. 35A).

The cell cycle status of CB CD34⁺ cells was analyzed in 5 indepenentexperiments with cells cultured for 3-13 days with either Dl-FRIL or acytokine combination (SCF, Flt3-L, G-CSF, IL-3, IL-6) (Bhatia et al., J.Exp. Med. 186:619-624, 1997; Conneally et al., Proc. Natl. Acad. Sci.USA 94:9836-9841, 1997). The percentage of cycling cells in SG₂M phasein both cultures decreased by half from day 3 to day 13, from 17.7% to9.1% for Dl-FRIL and from 50.9% to 23.2% for cytokines (Table 1) (3.4%of cells initially seeded were in SG₂M). TABLE 5 Percentage and numbersof cycling CD34^(±) cells (in SG₂M phase) cultured with Dl-FRIL orcytokines for 3, 6, 10 or 13 days Cyto- Fold Cyto- Dl-FRIL kinesincrease Days in Dl-FRIL kines (cells × (cells × induced by culture (%)(%) 10³) 10³) cytokines day 3 17.7 ± 1.9  50.8 ± 1.4  35 212 6 day 6 20± 2  26 43.6 389.5 9 day 10  10 ± 1.9 29.7 ± 1   26.4 553 21 day 13 9.1± 4.1 23.14 11.4 664.6 60CD34⁺ cells were cultured (2 × 10⁵ cells/0.5 ml) with Dl-FRIL or SCF +Flt3 ligand + IL-3 + G-CSF + IL-6.Cell cycle analysis was performed with propidium iodide staining.Cell numbers were calculated by multiplying percent of cycling cells bytotal cell numbers.Data represents mean ± SE values, from 5 experiments.

More dramatically, the number of cells in SG₂M phase during culturediffered from the 6.8×10³ cells in SG₂M phase initially seeded. Theaverage number of cycling cells in Dl-FRIL cultures remained relativelyconstant from 35×10³ cells at day 3 and 43.6×10³ cells at day 6 to areduced level of 26.4×10³ cells at day 10 and 11.4×10³ cells at day 13.This pattern of cells in cycle explains the low levels of total cellnumbers and progenitor cells (see FIG. 27). In contrast, the expectednumber of cycling cells in cytokine cultures increased dramatically. Theaverage number of cells in SG₂M phase in cytokine cultures increased by31-fold to 212×10³ cells at day 3, by 57-fold to 389.5×10³ cells at day6, by 81-fold to 553×10³ cells at day 10, and by 97.7-fold to 664.6×10³cycling cells at day 13, compared to 6.8×10³ cells in SG₂M seeded inculture (Table 1). Taken together, these results support the notion thatDl-FRIL preserves more immature progenitor cells in a quiescent state upto 2 weeks in culture compared to cytokine treated cultures.

To test whether Dl-FRIL acts in a dominant manner to prevent cytokinesfrom inducing the quiescent progenitor population into SG₂M phase, thecell cycle status of cells cultured with either Dl-FRIL or Flt3-L, inthe presence and absence of cytokines was analyzed, after 3 days ofsuspension culture as indicated in FIGS. 35A-35G and Table 6. FIGS.35A-35G show representative cell cycle histograms of CD34 ⁺ cellscultured with no addition (FIG. 35A), Dl-FRIL alone (FIG. 35B) orvarious cytokines or combinations thereof (FIGS. 35C-35G) for 3 days,and the mean percentage of cells in each cell cycle phase is summarizedin Table 6. TABLE 6 Cell cycle status of CD34+ cells, ex vivo culturedfor 3 days Culture conditions Sub G₀/G₁ G₀/G₁ SG₂M Dl-FRIL 9.4 ± 2.872.9 ± 2.8  17.7 ± 1.9  Flt3-L 9.4 ± 3.3 68.4 ± 2.1* 22.2 ± 5.4*Dl-FRIL + Flt3-L 8.6 ± 3.9 67.1 ± 1.8*  24.3 ± 5.65* Cytokines 2.6 ± 1.746.5 ± 4.5* 50.9 ± 1.4* Cytokines + Dl-FRIL 2.5 ± 0.6 47.5 ± 2.2* 50.0 ±2.7* Cytokines + Flt3-L 2.1 ± 0.5 47.7 ± 2.4* 50.2 ± 2.9*Data shown is mean percentage ± SE, from 4 independent experiments.p values = (vs. Dl-FRIL): *p < 0.05.

As was also shown in Table 5, Dl-FRIL maintains a significantly higherpercentage of quiescent cells and lower percentage of cycling cells(FIG. 35B and Table 6) compared to stimulation with cytokines (FIGS.35C-35G and Table 6). Cultures with Flt3-L alone led to a modest,although significant, decrease percentage of cells in G₀/G₁, (p<0.05)and to a corresponding increase in SG₂M phase, compared to cellscultured with Dl-FRIL alone (Table 6). Cultures containing Dl-FRIL andFlt3-L together slightly increased the percentage of cells in SG₂Mphase, from 17.7% (with Dl-FRIL alone) to 24.3% (p<0.05, Table 6). Asshown in Table 5, exposure of CB CD34⁺ cells to cytokines for 3 daysinduced a substantially greater proportion of cells into SG₂M phasecompared to culture with Dl-FRIL alone (p<0.05). The percentage of cellsundergoing apoptosis, as determined by subG₀/G₁ population, whencultured in Dl-FRIL was slightly higher than under cytokine conditions.Neither Dl-FRIL nor Flt3-L, under these culture conditions, effectedcytokine induction into SG₂M phase of the mostly quiescent CD34⁺ cellpopulation (Table 6).

EXAMPLE 7 Dl-FRIL Preserves CB Progenitors in a Dormant State in thePresence of Potent Stimulators

Dl-FRIL was purified from Dolichos lab lab as described above inExample 1. Dl-FRIL was assayed over a 5-log dose range (10 ng/mL-1,000ng/mL) on human CB MNC cultured in serum-defined medium containingagar-leukocyte conditioned medium, a potent source of a broad range ofstimulators. The number of viable MNC and progenitors were evaluatedafter 5 days in culture. Results from 1 of 3 representative experimentsare shown in Table 7 below. The number of Viable MNC at the end ofculture was reduced by 1.7- to 5-fold in cultures containing 10-1,000ng/ml of Dl-FRIL. The frequencies of myeloid and erythroid progenitorsin these cultures increased from 1.4- to 2.4-fold over the same doserange. TABLE 7 Progenitor frequency Dl-FRIL MNC (Colonies/10⁻⁵ MNC)(ng/ml) (×10⁻⁴) Myeloid Erythroid 1,000 20 90 +/− 85 150 +/− 42  100 5544 +/− 15 85 +/− 12 10 70 77 +/− 12 103 +/− 6  1 120 16 +/− 2  38 +/− 7 0.1 115 21 +/− 7  50 +/− 7  0 120 38 +/− 7  63 +/− 32Culture of CB MNC in DI-FRIL results in fewer MNC and a greaterfrequency of progenitors. 4 × 10⁶ CB MNC were cultured in 2 mL of AIMV(Life Technologies) containing Agar-SCM (StemCell Technologies) andvarying concentrations of Dl-FRIL.# Cultures were harvested after 5 days and the number of viable MNC andprogenitors were assessed. The colony data shown were normalized as thefrequency of progenitors per 10⁵ MNC.

A reduction in cell number and a corresponding increase in the frequencyof progenitors in cultures containing Dl-FRIL in the presence of potentstimulators are consistent with our hypothesis that Dl-FRIL can preventcytokine-induced proliferation and differentiation of progenitors. Table8 below shows the relative decrease of MNC in Dl-FRIL-containingcultures compared to controls and the relative increase in progenitorfrequency of progenitors. The total number of progenitors was reduced asexpected (progenitors are at varying stages of differentiation

no direct correlation between progenitor number and MNC would beexpected). TABLE 8 Fold Fold INCREASE Dl-FRIL DECREASE Progenitorfrequency (ng/ml) MNC Myeloid Erythroid 1,000 5.0 2.4 2.4 100 2.0 1.21.4 10 1.7 2.1 1.6 1 1.0 0.4 0.6 0.1 1.0 0.6 0.8 0 1.0 1.0 1.0Reduction in MNC correlates with the relative increase in frequency ofprogenitors. The relative reduction in MNC of Dl-FRIL cultures isconsistent with increased progenitor frequency in corresponding samples.

EXAMPLE 8 Dl-FRIL Protects CB MNC from the Toxicity of ChemotherapyDrugs

Experiments showing that Dl-FRIL can prevent proliferation anddifferentiation of CB progenitors in cultures containing potentstimulators indicated that Dl-FRIL protects progenitors from thetoxicity of cell cycle-active chemotherapy drugs. The culture systemdescribed above was adapted to a 96 well plate format and the widelyused chemotherapeutics, cytarabine (Ara-C) (FIG. 36A), doxorubicin (Dox)(FIG. 36B), and 5-fluorouracil (5-FU) (FIG. 36C) over a 5-log dose rangewere assayed on CB MNC cultured in the presence and absence of Dl-FRIL(see FIGS. 36A-36C). Dl-FRIL was purified from Dolichos lab lab asdescribed above in Example 1. Cultures containing Dl-FRIL (either at 10ng/ml or 100 ng/ml) resulted in a 2-3 log dose shift of CB MNC to Ara-C(FIG. 36A and Dox (FIG. 36B). As shown in FIG. 36C, the presence ofDl-FRIL in 5-FU cultures increased viability over a large dose range.Differences between the dose shift of Ara-C and Dox by Dl-FRIL comparedto 5-FU may be explained by recent reports demonstrating that 5-FU actsvia an RNA mechanism rather than as a DNA-specific drug (Bunz et al., J.Clin. Invest. 104:263-269, 1999).

EXAMPLE 9 Mice Tolerate High Levels of Dl-FRIL

The in vivo toxicity of Dl-FRIL was determined in mice. Initially,Dl-FRIL was administered intravenously to mice over a 3-log dose rangeof 0.006-1 mg/kg (0.32-20 μg/mouse). Dl-FRIL was well tolerated andthese mice have subsequently received 2 monthly challenges of Dl-FRILwithout any observable short- or long-term adverse effects.

Protocols to test chemoprotective properties of cytokines in micetypically involve daily pre-treatment (bolus or continuous delivery)regimens of 4-10 days before starting chemotherapy. Using this frameworkas a starting point, Dl-FRIL was injected at 5 mg/kg (100 μg/mouse)intravenously daily for 4 days. No gross adverse effects have beenobserved in over 150 mice treated with this dose regimen. Dl-FRIL waspurified from Dolichos lab lab as described above in Example 1.

The upper limits of Dl-FRIL toxicity were next explored by injecting asingle bolus intraperitoneal injection of Dl-FRIL (to accommodate 1 mLvolume) at 500 mg/kg and monitored the survival of mice for 48 hours. Ofthe four BALB/c mice receiving this treatment (2 males and 2 females,aged 5 months), only 1 mouse (a male) survived 48 hours. The survivingmouse's weight decreased by approximately 15% in the first 2 day andreturned to normal by day 4. The surviving mouse's blood counts were inthe normal range 3 days after injection of FRIL. The results demonstratethat even a very large dose of Dl-FRIL is not completely toxic.

Additional dose range finding studies are performed to determinetoxicity in mice receiving high doses of Dl-FRIL administered by variousroutes (intravenous, intraperitoneal, subcutaneous, and oral).

EXAMPLE 10 in vivo Modulation of Progenitors by Dl-FRIL in Mice

Using the initial planned dose regimen of Dl-FRIL (5 mg/kg×4 days) fortesting chemoprotection, hematopoietic parameters were examined in mice3, 5, and 7 days after completing Dl-FRIL treatment. Dl-FRIL waspurified from Dolichos lab lab as described above in Example 1.Weight-matched BALB/c mice (females, aged 8 weeks) were injectedintravenously with either 0.2 ml of Dl-FRIL (500 mg/ml) or 0.2 ml ofHBSS daily for 4 days. Two mice from each group were evaluated at 3days, 5 days, and 7 days after completing Dl-FRIL treatment. Blood wascollected in heparinized tubes by eye bleeds prior to sacrificing miceby CO₂. Two femurs and a spleen were harvested from each mouse and thesamples were processed within 1 hour. Progenitors wee accessed usingstandard hematopoietic colony assays (StemCell Technologies). Theresults from this study are shown below in Table 9. TABLE 9Hematopoietic parameters 3, 5, and 7 days after Dl-FRIL treatment (5mg/kg × 4 days) Cellularities Days RBC WBC BM Spleen 3 0.58 0.34 0.591.01 5 1.26 0.98 0.75 0.72 7 0.95 0.82 0.91 1.12 CFU-C CFU-E BFU-E + MixDays Freq. Total Freq Total Freq Total Bone marrow progenitors 3 1.851.10 1.63 1.01 1.61 1.01 5 1.17 0.87 0.86 0.63 1.03 0.80 7 1.81 1.690.61 0.57 2.59 2.47 Spleen progenitors 3 — — 2.83 3.35 — — 5 2.41 1.680.95 0.63 2.12 1.42 7 — — 1.74 1.78 — —Dl-FRIL data are reported as relative to control values.

As shown in Table 9, the peripheral blood counts (red blood cell (RBC)and white blood cell (WBC)) of Dl-FRIL-treated mice were found to bereduced by 1.7- and 2.9-fold, respectively, at 3 days, and returned tonormal by day 5. Bone marrow (BM) cellularity was also reduced by2.5-fold at day 3, and returned to normal after 7 days. The spleencellularity was lower at day 5 but normal at day 3 and day 7.

As shown in Table 9, the frequency of progenitors was slightly increasedin bone marrow, by 1.6- to 1.85-fold at day 3, but the total number ofprogenitors in the bone marrow remained unchanged. In the spleen, acompensatory organ during hematopoiesis stress, a 2.83-fold higherfrequency and 3.35-fold higher number of erythroid progenitors wereobserved in the spleen at day 3 (see Table 9). The frequencies and totalnumber of progenitors in the bone marrow appeared normal at day 5 butfewer mature erythroid progenitors (CFU-E) and more primitiveprogenitors (BFU-E/mix) were observed at day 7. A similar reduction inCFU-E was observed in spleens at day 5; otherwise, the frequencies andtotal numbers of progenitors increased from days 3-7.

EXAMPLE 11 DL-FRIL protects mice from 5-FU induced death in the criticalfirst week

A dose regimen was established to determine whether FRIL protects micefrom death resulting from hematopoietic toxicity of Ara-C and Dox. Thismurine 5-FU chemoprotection model is based on studies showing that asingle dose of 5-FU (150 mg/kg) resulted in >90% reduction of bonemarrow cellularity but had limited cytotoxic effect on stem cells(Lerner and Harrison, Exp. Hematol. 18:114-118, 1990). This finding wasconsistent with the understanding that stem cells reside in the bonemarrow in predominantly a quiescent state. Bone marrow cellularity inthese mice was restored after 2 weeks by the recruitment of the dormantprogenitors and responsive stem cells that escaped toxicity of 5-FU.Administration of a second dose of 5-FU (also at 150 mg/kg) 3

7 days after the initial dose killed those stem cells and progenitorsrecruited into S-phase in response to the first treatment of 5-FU(Lerner and Harrison, supra).

de Haan et al. (Blood 87:4581-4588., 1996) applied this model to testwhether prophylactic treatment of mice with hematopoietic regulators,pegylated SCF+IL11, could expand the stem cell and primitive progenitorcompartments and better protect mice from death by 5-FU inducedhematopoietic toxicity. These experiments demonstrated that althoughSCF+IL11 pretreatment could accelerate hematopoietic recovery after itwas underway by 4 days

5 days when compared to controls (from approximately 11 days to 7 daysfor 40% survival, the SCF+IL11 cytokine pretreatment strategy did notrescue mice from death when the second dose of 5-FU was administered inthe critical first week when stem cells are ablated (de Haan et al.supra).

In this study described below, the pretreatment dose regimen for Dl-FRILdescribed above (5 mg/kg×4 days) was selected based on the requirementof treating animals with cytokines for several days prior to startingchemotherapy and because Dl-FRIL-treated mice easily tolerated doses (5mg/kg) that were 10- to 100- fold greater than that used for cytokines.Since FRIL and cytokines act on progenitors in the same concentrationrange (ng/ml), this pretreatment dose regimen was used to test whetherDl-FRIL can protect mice from 5-FU administered at 2 intervals in thefirst week: 5-FU (150 mg/kg) was injected at day 0 and then a seconddose (also at 150 mg/kg) was injected either on day 3 (dO/3 doseinterval) or day 5 (dO/5 dose interval). No survival on the day 3interval was observed by de Haan et al. (supra) at day 3.

Dl-FRIL was purified from Dolichos lab lab as described above in Example1.

Weight-matched BALB/c mice (10 mice/group) were injected intravenouslywith either with 0.2 mL of Dl-FRIL (500 mg/mL) or 0.2 mL of HBSS dailyfor 4 days. Two hours after the final treatment of Dl-FRIL, mice wereinjected intraperitoneally with 5-FU (150 mg/kg). Groups of micereceived a second dose of 5-FU (150 mg/kg) at either day 3 or day 5. Nomice died from a single dose of 5-FU.

As shown in Table 10, Dl-FRIL pretreatment improved survival of mice intwo separate experiments. TABLE 10 5-FU Dose Interval D0/3 D0/5 Exp.Mice FRIL HBSS FRIL HBSS 1 Males, 16 wk 3/10 0/10 N.T. N.T. 2 Females, 8wk 0/10 0/10 4/10 1/10Improved survival of mice pretreated with FRIL (5 mg/kg × 4 days) priorto undergoing 5-FU dose intervals of d0/3 and d0/5.

In the first experiment, 3 of 10 mice survived a d0/3 dose interval of5-FU compared to no mice in the HBSS pretreatment control. In the secondexperiment, 4 of 10 mice pretreated with Dl-FRIL survived a d0/5 doseinterval of 5-FU compared to 1 of 10 for HBSS pretreated mice.

EXAMPLE 12 Optimization of the Dose Regiment of a FRIL Family Member toProtect Mice from 5-Fu Induced Death

FRIL family members are relatively abundant in legumes. For example,Dl-FRIL accounts for approximately 0.02% of the mass of hyacinth beans.

Dl-FRIL is purified by carbohydrate affinity chromatography as describedin Example 1, and is evaluated for purity by SDS-PAGE (5 discrete bandsare visualized on an overloaded gel; see FIG. 37); is analyzed for massand composition by amino acid analysis; and is assayed in the cord bloodprogenitor assay described above.

The murine 5-FU chemoprotection model (Lemer and Harrison, supra; deHaan, supra) is used to empirically derive the optimal dose regimen ofDl-FRIL to protect mice from death. Dl-FRIL is administeredintravenously to mice over a 3-log dose range (5-5,000 mg/kg) undervarious regimens that include Dl-FRIL treatment prior to chemotherapy(daily from 3 days to 2 hour before initiation of chemotherapy) andduring chemotherapy.

The mice used in these studies are BALB/c female mice, 8-10 weeks atoutset of experiments Jackson Laboratory, Bar Harbor, Me.), weightmatched each for experiment, where there are 10 mice per group. Organsfrom mice receiving a dose of 5,000 mg/kg of FRIL (with no 5-FUtreatment) are collected for toxicity studies

The five doses of Dl-FRIL are 0, 5, 50, 500, and 5,000 μg/kg. The fourdose regimens of Dl-FRIL will be -2 hour; -1 day and -2 hour; -2 day, -1day, and -2 hour; -3 day, -2 day, -1 day, and -2 hour prior to 5-FUtreatment. The two maintenance regimens are either daily×7 day (-2hourto day 7) or every other d (days 0, 2, 4, 6). Thus, one group of micewill receive a dose of Dl-FRIL daily for 7 days; while the second groupwill receive a dose of Dl-FRIL every other day for 7 days.

The 5-FU dose intervals of 150 mg/kg are at dose intervals of d0/3,d0/5, and d0/7.

EXAMPLE 13 A FRIL Family Member has Chemoprotective Properties withWidely used Cell Cycle-Active Chemotherapeutics

After establishing the optimal dose regimen of a FRIL family member, theFRIL family member's ability to protect mice from death by cytarabine(Ara-C) and doxorubicin.

Initial dose regimens of cytarabine (Ara-C) and doxorubicin are asfollows: Doxorubicin

14 mg/kg as single bolus i.p. injection (Grzegorzewski et al.,J.Exp.Med. 180:1047-1057, 1994); Ara-C-300 mg/kg at time as an i.p.injection at 0 and 12 hours (Paukovits et al., Blood 77:1313-1319,1991). Further studies are based on targeted clinical indication

EXAMPLE 14 Characterization of the Hematopoietic Status of Mice DuringOptimal Dose Regimen of a FRIL Family Member to Protect Mice from 5-FUInduced Death

Peripheral blood counts and the status of hematopoietic progenitors(frequency, total number, and cycling status) are characterized in miceduring and after receiving the optimal dose regimen of a FRIL familymember.

To do this, mice injected with a dose regimen of a FRIL family memberwith no 5-FU treatment are evaluated daily during and for one week aftertreatment with the FRIL family member. The mice are evaluated for thefollowing hematopoietic parameters: WBC and RBC counts; bone marrow andspleen cellularities; and progenitor status, which includeshematopoietic colony assays (StemCell Technologies). The progenitorsassayed are myeloid (CFU-C), erythroid (CFU-E), and primitive,multipotential (BFU-E/Mix). The frequencies and total numbers aredetermined, as well as the cycling status of these cells, as measured by³H-thymidine suicide assay (Moore et al., Exp. Hematol. 14:222-229,1986).

EXAMPLE 15 Analysis of Pharmacology and Toxicology of FRIL in Mice

The clearance of a FRIL family member from the circulation and itsaccumulation in the body is determined by preliminary pharmacokinetics.

For these studies, ¹²⁵I-FRIL is injected into mice (dosage of FRIL basedon optimization results). Clearance of FRIL from the blood is evaluatedat the following timepoints after injection: 5 min., 15 min., 30 min., 1hour, 2 hours, 4 hours, 8 hours, 12 hours, 36 hours, 48 hours. Two miceare evaluated at each timepoint. Following this study, the animals aresacrificed and the organs collected and evaluated.

Dose-range finding experiments determine the maximal tolerated dose of aFRIL family member by various routes of administration (intravenous,intraperitoneal, subcutaneous, and oral). To do this dose range findingstudy, four routes at four doses are used. The routes of administrationare intravenous, intraperitoneal, subcutaneous, oral. The highest doseof a FRIL family member is 1 g/kg. The dosage of the FRIL family memberis reduced by 2-fold until mice survive. Survival is measured at 48hours after treatment. Other clinical observations are made, includingbehavorial, lethargy, vocalization, diarrhea. CBC analyses are made. Theanimals are evaluated for any necropsy (i.e., gross lesions). Followingthis study, the animals are sacrificed and the organs collected andevaluated.

Acute dose toxicity studies allow identify target organs that maydevelop lesions after exposure to a FRIL family member. For these acutedose toxicity studies, a dose is selected, and a FRIL family member isinjected daily for 7 days. Acute toxicity is evaluated at 7 days, andrecovery from acute toxicity is evaluated at 21 days. Blood chemistries,target organs, bone marrow and blood, and other health indicator areevaluated.

Hypersensitivity studies in guinea pigs are performed to test for anyadverse immunologic reactions. To do this, fifteen guinea pigs (5 FRIL,5 DNCB positive control, 5 saline negative control) are used. A FRILfamily member is intradermally injected at 0.1 mL. Daily clinicalobservations at site for redness and edema are compared to the DNCBpositive control. The FRIL guinea pigs are challenged at at 2 weeks with0.05 mL of the FRIL family member, and daily clinical observations aremade.

A determination of development of mouse anti-FRIL family memberantibodies in mice receiving treatment with a FRIL family member is madeto determine the extent and nature of the body's response to a FRILfamily member. To do this, a FRIL family member is attached to Dynal'stosylactivated magnetic beads. The FRIL family member-coated beads areincubated with plasma (pooled or individual) from a mouse who hasreceived treatment with the FRIL family member. Using rabbit and ratantiserum to FRIL used as positive control, the presence of FRIL familymember-specific antibodies is evaluated by SDS-PAGE and Western blotanalysis (horseradish peroxidase or chemiluminesce). Lastly, adetermination is made as to whether sugar blocks antibody binding(a-D-mannopyranoside and negative control).

EXAMPLE 16 Purification of Progenitor Cells using Dl-FRIL-CoatedMagnetic Beads

Using magnetic beads coated with a non-limiting FRIL family member,Dl-FRIL, a population of progenitor cells was isolated andcharacterized. To do this, the following methods were used.

Preparation of FRIL-Beads for Cell Isolation

Dl-FRIL was purified from Dolichos lab lab seeds as described inExample 1. Dl-FRIL can be immobilized on magnetic beads (M-280 DynabeadsTosylactivated, Lake Success, N.Y.) via amino- and sulfhydryl-groups ofthe lectin according to the manufacturer's directions. Dl-FRIL can alsoimmobilized on magnetic beads by a biotin-strepavidin interaction.

In this example, Dl-FRIL was immobilized on magnetic beads by abiotin-strepavidin interaction. Biotinylation of Dl-FRIL via primaryamine-groups (EZ-Link Sulfo-NHS-LC-LC-Biotin, Pierce Chemical Company,Rockford, Ill.) was carried out according to the manufacturer'sdirections. Biotinylated Dl-FRIL was incubated with strepavidin-labeledmagnetic beads (Dynal or Miltenyi Biotec, Auburn, Calif.) according tothe manufacturer's directions.

Preparation of Cells

Human cord blood (CB), peripheral blood, and bone marrow, collected insterile receptacles containing anticoagulant (e.g., heparin, EDTA), wasprocessed to isolate mononuclear cells (mnc) within six hours ofcollection by density centrifugation on Ficoll-Paque PLUS (PharmaciaBiotech, Piscataway, N.J.) according to the manufacturer's directions.Mononudear cells harvested at the interface of plasma and Ficoll-Paquewere washed and resuspended in serum-defined medium (e.g., XVIVO-10,Biowhittaker, Walkerville, Md. or AIM-V, Life Technologies, Rockville,Md.).

Dl-FRIL-Bead Cell Isolation

Dl-FRIL-coated beads specifically bound a minor mnc population found inCB, peripheral blood, and bone marrow. A ten-fold excess ofDl-FRIL-beads was incubated with the cells. For CB, where Dl-FRIL-beadscaptured approximately 1% of mnc, the ratio of beads to cells was 1:10,or 10-fold greater number of beads for every target cell. The ratio forother FLT3-expressing cell populations, was hematopoietic andnon-hematopoietic, was experimentally determined by serial exposure ofcells to fresh Dl-FRIL-beads.

Dl-FRIL-beads were washed twice in serum-defined medium prior to use. Analiquot of Dl-FRIL-beads was added to 10 mL of serum-defined medium in a15 mL conical centrifuge tube (Falcon, Becton-Dickinson, Lincoln, N.J.),mixed, and placed in a magnet (Dynal or Miltenyi Biotec, depending onsource of magnetic beads) for ten minutes. Medium was aspirated with a10 mL pipette without disturbing beads bound to walls of centrifuge tubeby the magnet charge from the magnet. After washing, 0.5 mL ofserum-defined medium was added to the tubes to wash the beads from thewalls to the bottom of the conical tube. Medium was added to beads in asmall volume (<2 mL) and the centrifuge tube was tumbled on a rocker ina cold room (i.e., at approximately 4° C.) for one hour. Afterincubation, serum-defined medium was added to a final volume of 10 mLand the tube was placed in the magnet for ten minutes. Medium wasremoved by aspiration without disturbing cells bound to Dl-FRIL-beads onthe walls of the centrifuge tube via the magnetic charge. Cells werewashed a second time by removing the conical tube from the magnet,adding 10 mL of serum-defined medium, mixing cells, and placing theconical tube back onto the magnet. Following aspiration of the medium,the final volume was adjusted to 2 mL.

Detachment of Dl-FRILbeads from Cells

For some applications, detachment of Dl-FRIL-beads from cells ispreferred. About half of Dl-FRIL-beads detached from CB mnc afterovernight incubation on a rocker in the cold room. Although bindingstudies have demonstrated that excess mannose and α-methyl α-D-mannosideprevent Dl-FRIL from binding to Flt3, neither sugar released tightlybound Dl-FRIL-beads from CB mnc. To remove Dl-FRIL-beads from thissubpopulation of cells, the cells were incubated in 100 mM trehalose(Sigma, St. Louis, Mo.) for one hour on a rocker in the cold room.

It should be noted that since Miltenyi beads are very small(approximately 50 nm) as compared to Dynal beads (approximately 10 μm),when Miltenyi beads were used to purify Dl-FRIL-binding progenitorcells, the beads were allowed to remain attached to the purifiedprogenitor cells.

Receptor Tyrosine Kinase Gene Expression

Receptor tyrosine kinase gene expression was characterized by RT-PCR.

Using these methods, the following results were obtained:

Functional Properties of Dl-FRIL Bead-Selected CB mnc

The progenitor capacity of Dl-FRIL-selected CB mnc was tested in amethylcellulose colony assay under conditions to promote proliferationand differentiation along either the hematopoietic or endotheliallineages (as described above. Table 11 shows the number of hematopoieticcolonies (myeloid, erythroid, and mix) and endothelial colonies (other)that formed after culture of unselected cells (CB mnc), Dl-FRIL-selectedcells (Dl-FRIL⁺) and CB mnc that did not bind to Dl-FRIL-beads(Dl-FRIL⁻). TABLE 11 Response of Dl-FRIL-selected cells to hematopoieticand endothelial stimuli Stimulator Myeloid Erythroid Mix Other Total+CSFs CB mnc 1 3 2 0 6 Dl-FRIL⁺ 14 10 4 0 28 Dl-FRIL⁻ 1 1 2 0 4 +VEGF CBmnc 0 0 0 2 2 Dl-FRIL⁺ 0 0 0 19 19 Dl-FRIL⁻ 0 0 0 5 5Cord blood mononuclear cells were isolated by Ficoll-Paque, beadselected, and plated in MethoCult□, StemCell Technologies, Vancouver,BC, Canada).

As shown in Table 11, Dl-FRIL-selection increased the number ofhematopoietic colonies (stimulated with colony stimulating factors(CSFs) by 14-fold for myeloid colonies, 3.3- to 10-fold for erythroidcolonies (CB mnc and Dl-FRIL- cells, respectively), and by 2-fold formixed colonies. Similar levels of Dl-FRIL-bead enrichment was observedfor endothelial colonies (stimulated with vascular endothelial growthfactor (VEGF)): 9.5-fold over CB mnc and 3.8-fold for Dl-FRIL⁻ cells.

Cell Surface Phenotypic Properties of Dl-FRIL Bead-Selected CB mnc

The cell surface phenotypic properties of Dl-FRIL bead-selected CB mncwas characterized by flow cytometry. Table 12 shows the phenotypes (bypercentage of cells expressing the indicated cell surface phenotypemarker) of the three CB cell populations: (1) cells not selected byDl-FRIL-beads (Dl-FRIL⁻); (2) cells that detached from Dl-FRIL-beadsafter overnight incubation in the coldroom on a rocker (Dl-FRIL⁺); and(3) cells that retained Dl-FRIL-beads after overnight incubation(Dl-FRIL⁺⁺). The two Dl-FRIL-binding cell populations were analyzedseparately to see whether tightness of binding (avidity) related to typeof cells selected. Isotype control levels were set at 2%; all values of2% represent no reactivity with test antibody. TABLE 12 Flow cytometricanalysis of Dl-FRIL-selected CB mnc Dl-FRIL⁻ Dl-FRIL⁺ Dl-FRIL⁺⁺ AntigenCell Type (%) (%) (%) CD3 Mature T 26 35 6 CD11b Mac-1, CR3 19 35 67CD11c LeuCAMc 10 22 32 CD13 Pan myeloid, CFU-GM 5 <2 <2 CD19 Pan B 4 512 CD32 Low affinity IgG Fcγ-R 5 19 26 CD33 Myeloid progenitors 3 2 8CD34 Pan progenitors <2 <2 <2 CD38 Activated T 88 96 93 CD42a PlateletgpIX 5 2 7 CD69 Early activation ag 6 8 14 (EA-1) CDw90 Thy-1,progenitor subset 8 14 13 CD117 c-kit, progenitors 4 2 2

As shown in Table 12, Dl-FRIL-beads did not capture CB mnc that expressCD34, the hallmark marker of hematopoietic stem cells and progenitors.This observation was unexpected because Dl-FRIL-selected cells enrichfor progenitors (see Table 11). Although CB CD34⁺ cells uniformlyexpress FLT3, only 70% of Flt3⁺ CB mnc also express CD34 (Rappold etal., Blood 90:111-125, 1997). Consequently, 30% of CB mnc expressed thephenotype of CD34⁻Flt3⁺. Dl-FRIL-beads appeared to capture this latterpopulation of cells.

Cells expressing dendritic cell (DC) markers, CD11b and CD11c, wereenriched approximately 2-fold in the Dl-FRIL⁺ cell population and over3-fold in the Dl-FRIL⁺⁺ cell population (Table 12). This observation isconsistent with reports that Flt3 is involved in dendritic cellproliferation and maturation in mice (Pulendran et al., J. Immunol.159:2222-2231, 1997) and humans (Miller et al., Blood 93:96-106., 1999).The rare hematopoietic dendritic cell population is useful in inducingtumor regression and for the treatment of AIDS.

Differences of the cell surface phenotypes were observed betweenDl-FRIL⁺ and Dl-FRIL⁺⁺ CB cells (see Table 11). The percentage of CD3 Tcells decreased from 35% for Dl-FRIL⁺ cells to 6% for Dl-FRIL⁺⁺ cells.Conversely, the percentage of CD11b⁺ cells and CD11c⁺ cells increasedfrom 35% to 67% and from 22% to 32% for Dl-FRIL⁺ and Dl-FRIL⁺⁺ cellpopulations, respectively.

Cells that retained Dl-FRIL-beads after overnight incubation on a rockerin the cold room (Dl-FRIL⁺⁺ cells) were observed as single cells or asclumps of bead-bound cells. These clumps could not be disrupted eitherby mechanical means or by elution with competing sugars, mannose ormannose derivatives (data not shown). From studies to characterize thecarbohydrate-binding properties of Dl-FRIL, α,α-trehalose demonstrated a3.6-fold greater potency than mannose and a 1.6- to 2.1-fold greaterpotency than α-methyl α-D-mannoside derivatives that were tested (Mo etal., Glycobiology 9:173-179,1999). Incubation of clumped Dl-FRIL-beadbound cells with 100 mM Trehalose effectively disrupted the clumpedcells and removed most of the Dl-FRIL-beads from cells.

Two populations of trehalose-disrupted Dl-FRIL⁺⁺ cells were analyzed byflow cytometry: cells that no longer bound beads (Dl-FRIL⁺⁺) and cellsthat still retained beads following incubation with trehalose(Dl-FRIL⁺⁺⁺). The results of one experiment are shown in Table 13. TABLE13 Flow cytometric analysis of Dl-FRIL-selected CB mnc after exposure totrehalose Dl-FRIL⁺⁺ Antigen Cell Type (%) Dl-FRIL⁺⁺⁺ (%) CD3 Mature T 63 CD11b Mac-1, CR3 52 76 CD11c LeuCAMc 16 43 CD13 Pan myeloid, CFU-GM 7246 CD19 Pan B 5 9 CD32 Low affinity IgG Fc-γ 43 19 CD33 Myeloidprogenitors 56 15 CD34 Pan progenitors 2 2 CD117 c-kit, progenitors 2 12CD135 Flt3, progenitors 2 5

The difference in cell surface phenotypes between Dl-FRIL⁺⁺ cells andDl-FRIL⁺⁺⁺ cells in Table 13 was greater than those observed forDl-FRIL⁺ cells and Dl-FRIL⁺⁺ cells in Table 12. In Table 13, thepercentage of CD3, CD13, CD32, and CD33 cells decreased by 1.6- to3.7-fold in Dl-FRIL⁺⁺⁺ cells compared to Dl-FRIL⁺⁺ cells. Conversely,the percentage of CD11b, CD11c, CD19, CD117, and CD135 cells increasedby 1.5- to 6-fold in Dl-FRIL⁺⁺⁺cells compared to Dl-FRIL⁺⁺ cells. Againno CD34 was observed in either cell population.

The increase in percentage of cells that express CD117 and CD135, twotyrosine kinase receptors central to hematopoietic stem cell andprogenitor function (Lyman and Jacobson, Blood 91:1101-1134, 1998),suggested that avidity of Dl-FRIL-bead binding of cells might correspondto the primitive status of the cells. The studies described herein whichcharacterize the carbohydrate binding properties of Dl-FRIL supportedthis notion. Dl-FRIL has neither an extending carbohydratecombining-binding site nor a hydrophobic binding site adjacent to it (Moet al., Glycobiology 9:173-179, 1999). Dl-FRIL binds most tightly to atrimannoysl structure that is the basis for N-linked glycosylation inmammals. Consequently, Dl-FRIL may bind cells that have undergone lessprocessing of glycosylation, which is consistent with more primitivecells. This property of Dl-FRIL-binding may provide a unique method toisolate primitive cells not currently possible by antibodies to CD34.Thus, Dl-FRIL binds the normally glycosylated FLT3 receptor more tightlythan the FLT3-Ligand binds to normally glycosylated FLT3. Dl-FRIL bindsnormally glycosylated FLT3 receptor more tightly than the typicalantibody binds its specific ligand.

Receptor Tyrosine Kinase Gene Expression in Dl-FRIL-Selected CB Cells

The number of cell surface receptors and markers increases aspluripotent hematopoietic stem cells proliferate and differentiate. Thenumber of functional receptors on the most primitive cells is probablyless than ten. The levels of detection for flow cytometry are probablyin the range of several hundred cell surface molecules. Consequently,analysis of primitive cell populations cannot be analyzed by flowcytometry.

The presence or absence of functional tyrosine kinase receptors onprimitive cells was further characterized by RT-PCR. Expression of Flt3,Kit, Fms, Flk1, Flt1, and Flt4 mRNA was determined for CB mnc,CD34-selected cells, and Dl-FRIL-selected cells. For CB mnc, the PCRproducts for the receptors were either faint or not detectable. Thepattern of gene expression for cells selected by CD34-beads orDl-FRIL-beads was the same; all tyrosine kinase receptors showedstronger PCR signals. These data suggest that receptors associated withstem cells (Flt3 and Kit) and primitive endothelial cells (FLk1, FIt1,and Flt4) are also detected in Dl-FRIL selected cells.

EXAMPLE 17 Use of Beads Coated with a FRIL Family Member to IsolateCD34⁻ Primitive Stem Cells

A rare human stem cell population with the phenotype of CD34⁻CD38⁻Lin⁻has been identified by its ability to establish multilineage engraftmentin NOD/SCID mice (Bhatia et al., Nat. Med. 4:1038-1045, 1998). Theserepopulating cells give rise to stem cells that express the hallmarkCD34 marker. Isolating CD34⁻CD38⁻Lin⁻ cells is labor-intensive methodsof negative selection that includes the use of immunomagnetic beads andflow cytometry to deplete cells that express CD34, CD38, and lineagemarkers. Rapid, efficient, positive selection of CD34⁻CD38⁻Lin⁻ cellswould be preferable for clinical uses. However, the absence of thehighly expressed CD34 marker and low number of functional receptors onthis rare population of cells will prevent use of antibodies for cellisolation.

FRIL attached to magnetic beads are used in a unique method to isolatethe rare CD34⁻CD38⁻Lin⁻ cell population by binding primitive cells thatexpress this phenotype. Isolation of CD34⁻CD38⁻Lin⁻ is achieved by asingle-step cell isolation. However, since FRIL-beads also recognizecells that express CD11b, CD11c, and CD38, optimal isolation ofCD34⁻CD38⁻Lin⁻ cells is improved by first negatively selecting unwantedcells by immunomagnetic beads that bind to CD11b, CD11c, and/or CD38.

EXAMPLE 18 Use of Beads Coated with a FRIL Family Member to IsolateNormal Stem Cells from Patients with Leukemia

A majority of leukemias express the phenotype of CD34⁺Flt3⁺ (Carow etal., Blood 87:1089-1096, 1996). Consequently, methods that rely on CD34expression cannot distinguish normal stem cells and progenitors fromleukemic cells in the bone marrow and peripheral blood of patients.

FRIL does not interact with two cell leukemic lines tested with the CD34⁺Flt3⁺ phenotype (KG₁-A and ML-1). FRIL neither effects growth of theseleukemic cell lines nor do FRIL-beads capture appreciable numbers ofcells (data not shown). Since FRIL-beads select normal progenitors withthe phenotype of CD34⁻Flt3⁺, FRIL-beads provide a unique method thatdistinguishes between normal and leukemic cells.

A FRIL family member attached to magnetic beads is used to isolatenormal hematopoietic stem cells and progenitors from the bone marrow andperipheral blood of leukemia patients. This is accomplished using amethod similar to leukopheresis, where blood is passed through a devicethat retains cells of interest. In this example, FRIL-beads bindsCD34⁻Flt3⁺ normal cells. Since FRIL also interacts with Flt3-expressingCD11b and CD11c cells, prior exposure and removal of the cells thatimmunomagnetic beads that bind to CD11b and/or CD11c (i.e., negativeselection) may permit enrichment of primitive cells.

EXAMPLE 19 Use of FRIL-Beads to Isolate Dendritic Progenitors and MatureCells from Normal Individuals

Dendritic cells (DC) are immune cells that capture antigens and initiateT cell-mediated immune responses (Banchereau and Steinman, Nature392:245-252, 1998). DC act as first lines of defense in the skin, gut,and lymphoid organs. Antigens on DC can activate naive and quiescent Tcells and small numbers of DC pulsed with lose dosages of antigensstimulate strong T cell responses. Under certain circumstances, DC alsoinduce T cell tolerance. Consequently, the unique properties of DC hasgenerated significant interest to use these cells to treat cancer andAIDS.

DC are derived from CD34⁺ progenitors in the bone marrow of humans. Thecytokines GM-CSF, TNF-α, and Flt3 ligand (FL) influence DC development(Banchereau and Steinman, supra; Pulendran et al., J. Immunol.159:2222-2231, 1997). Injection of FLT3-Ligand in mice dramaticallyincreases the number of DC (Pulendran et al., supra). FRIL interactswith the Flt3 receptor on DC, and FRIL-beads capture cells with thedendritic phenotype of CD11b and CD11c. Selecting dendritic cells withFRIL-beads from human bone marrow, peripheral blood, or cord bloodallows the efficient and effective isolation of DC for clinical use.

EXAMPLE 20 Use of FRIL-Beads to Isolate Endothelial Stem Cells andProgenitors

Endothelial stem cells and progenitors give rise to cells that formblood vessels in a process called angiogenesis. During strokes and heartattacks, new blood vessels are needed to repair damage. Activation ofendothelial stem cells and progenitors to produce more mature cells ismediated by the cytokines that activate the Flk1/KDR, Flt1, and Flt4tyrosine kinase receptors. Flt3 is expressed on very primitiveendothelial progenitors. FRIL-beads are used to capture a population ofcells from cord blood that express all of these receptors.

EXAMPLE 21 Use of FRIL Family Members and Non-FRIL Family Member Lectinsto Alter Signal Transduction and Other Cellular Pathways

Drugs designed to alter signal transduction pathways need tospecifically distinguish target cells. FRIL is used as a targetingvehicle to deliver small molecules to Flt3-expressing cells such as stemcells, progenitors, and dendritic cells. FRIL has several advantages fordrug delivery: 1) FRIL specific for Flt3; 2) FRIL is stable in thecytoplasm; 3) FRIL is capable of undergoing conjugation with smallmolecules; 4) FRIL can be delivered in dose-responsive manner; and 5)FRIL provides specificity for overlapping pathways of signaltransduction.

Other legume- or bulb-derived lectins can also delivery small moleculedrugs to specific cell populations. For example, the lectins PHA andConA both bind to CD3-T cell receptor complex and the FC-gamma receptor(CD32) (Leca et al. Scand. J. Immunol. 23:535-544, 1986); UDA binds theVβ domain of the T cell receptor (Galelli et al., J. Immunol.151:1821-1831, 1993).

Standard methods of conjugation are used to attach small molecules,oligos, or enzymes to plant lectins.

EXAMPLE 22 Purification and Cloning of YamFRIL from Sphenostylisstenocarpa

Dry seeds of Yam bean (Sphenostylis stenocarpa) were ground in a coffeemill and the powder was extracted with 5 volumes of 10 mM Na Acetatebuffer, pH 5.2, containing 1 mM CaCl₂ for 1 hour at 4° C. Aftercentrifugation, the clear supernatant was neutalized with Tris-HCl pH8.0.

YamFril purification was achieved through and absorption on a ovalbumingel affinity column (Sigma) and was eluted with 200 mM trehalose.

The resulting protein was fractionated into 2 polypeptides that weresubmitted to N-terminal amino acid sequences. Beta band: AQSVSFTFTKFDSDQ(SEQ ID NO: 9) Alpha band: AASNNVVAVEFDTXPN (SEQ ID NO: 10)

Reverse-transcriptase PCR was performed on total RNA obtained fromInternational Institute of Tropical Agriculture (Ibadan, Oyo State,Nigeria), using degenerate primers based on the alpha and betaN-terminus sequences (i.e., SEQ ID NO: 10 and SEQ ID NO: 9,respectively).

3′ RACE PCR was performed on total and polyA+RNA using gene specificprimers with an Anchor Primer.

Partial cDNA clones were obtained and the following sequences deduced.YamFril: partial mRNA sequenceACGAAGTTCGACAGCGACCAAAAGGATCTTATGTTCCAAGGTCATACCATTTCTAGCAGC (SEQ ID NO:7) AATGTCATACAACTCACCAAGTTAGACAGTAATGGAAACCCTGTGAGTACCAGTGTGGGAAGAGTGTTATACTCTGCACCATTGCGCCTTTGGGAAAGCTCTACAGTAGTGTCAACCTTTGAGACCACTTTCACCTTTCAAATCTCAACACCTTACACTAGTCCTCCTGGTGATGGGCTCGCCTTCTTCCTTGCACCATATGACACTGTCATCCCTCCAAATTCTGCTGGCAATCTTCTTGGACTCTTTCCTAACTTAAATGCTTTAAGAAACTCCACCACCAGTAAAGAAACCACTATTGATGTCAATGCTGCATCTAACAACGTTGTTGCCGTTGAATTTGACACCTACCCTAACGACAATATTGGTGATCCAAGATACAAACACATTGGAATCGATGTCAACTCTATCAGGTCCAAGGCAACTGTTGCGTGGGACTGGCAAAATGGGAAAACAGCCACTGCACACATCAGCTATAACTCTGCCTCTAAAAGACTATCTGTTACTACTTTTTATCCTGGGGGTAAAGCTGTGAGTCTTTCCCATGACGTTGAGCTCACTCAAGTGCTTCCTCAATGGATTAGAGTAGGGTTCTCTGCTTCAACAGGATTAGAGAAA YamFril: deduced amino acid sequenceAQSVSFTFTKFDSDQKDLMFQGHTISSSNVIQLTKLDSNGNPVSTSVGRVLYSAPLRLWE (SEQ ID NO:8) SSTVVSTFETTFTFQISTPYTSPPGDGLAFFLAPYDTVIPPNSAGNLLGLFPNLNALRNSTTSKETTIDVNAASNNVVAVEFDTYPNDNIGDPRYKHIGIDVNSIRSKATVAWDWQNGKTATAHISYNSASKRLSVTTFYPGGKAVSLSHDVELTQVLPQWIRVGFSASTGLEK

1-61. (canceled)
 62. A method for alleviating or reducing thehematopoietic progenitor cell-depleting activity of a therapeutictreatment in a patient, comprising administering to the patient atherapeutically effective amount of a pharmaceutical composition priorto administration of the therapeutic treatment to the patient, whereinthe pharmaceutical formulation comprises: (a) a pharmaceuticallyacceptable carrier; and (b) a protein that: (1) binds to a normallyglycosylated FLT3 receptor; (2) has at least 95% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3) preserveshematopoietic progenitor cells.
 63. The method of claim 62, wherein thepatient is human.
 64. The method of claim 63, wherein the patient hascancer.
 65. The method of claim 62, wherein the therapeutic treatment isselected from the group consisting of a radiotherapeutic, achemotherapeutic, or a combination of a radiotherapeutic and achemotherapeutic.
 66. The method of claim 65, wherein thechemotherapeutic is selected from the group consisting of cytarabine,doxorubicin, and 5-fluorouracil.
 67. The method of claim 62, wherein theprotein is a recombinant protein.
 68. A method for preserving progenitorcells ex vivo, comprising contacting a population of cells comprising atleast one progenitor cell with an effective amount of a pharmaceuticalcomposition for an effective period of time, wherein the progenitorcells in the population are rendered quiescent, wherein thepharmaceutical formulation comprises: (a) a pharmaceutically acceptablecarrier; and (b) a protein that: (1) binds to a normally glycosylatedFLT3 receptor; (2) has at least 95% amino acid sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO:8; and (3) preserves hematopoieticprogenitor cells.
 69. The method of claim 68, wherein the progenitorcells are from a human.
 70. The method of claim 68, wherein thepopulation of cells is bone marrow cells.
 71. The method of claim 68,wherein the non-progenitor cells in the population of cellsdifferentiate or die.
 72. The method of claim 68, wherein the populationof cells is removed from a cancer patient prior to treatment of thecancer patient with a therapeutic treatment having a hematopoieticprogenitor cell-depleting activity.
 73. The method of claim 72, whereinthe therapeutic treatment is selected from the group consisting of aradiotherapeutic, a chemotherapeutic, or a combination of aradiotherapeutic and a chemotherapeutic.
 74. The method of claim 73,wherein the chemotherapeutic is selected from the group consisting ofcytarabine, doxorubicin, and 5-fluorouracil.
 75. The method of claim 68,wherein the protein is a recombinant protein.
 76. A method forpreserving progenitor cells in vivo, comprising administering to apatient an effective amount of a pharmaceutical composition for aneffective period of time, wherein the progenitor cells in the patientare rendered quiescent, wherein the pharmaceutical formulationcomprises: (a) a pharmaceutically acceptable carrier; and (b) a proteinthat: (1) binds to a normally glycosylated FLT3 receptor; (2) has atleast 95% amino acid sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO:8; and (3) preserves hematopoietic progenitor cells.
 77. Themethod of claim 76, wherein the patient is human.
 78. The method ofclaim 77, wherein the patient is a cancer patient.
 79. The method ofclaim 76, wherein the effective amount of the pharmaceutical compositionis administered prior to the treatment of the patient with a therapeutictreatment having a hematopoietic progenitor cell-depleting activity. 80.A method for identifying a progenitor cell, comprising contacting acandidate cell with a FRIL family member molecule, wherein binding ofthe candidate cell to the FRIL family member molecule identifies thecandidate cell as a progenitor cell, wherein the FRIL family membermolecule comprises a protein that: (1) binds to a normally glycosylatedFLT3 receptor; (2) has at least 95% amino acid sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO:8; and (3) preserves hematopoieticprogenitor cells.
 81. The method of claim 80, wherein the candidate cellis in a population of cells.
 82. The method of claim 80,wherein thecandidate cell is from a human.
 83. A progenitor cell identified by themethod of claim
 80. 84. A method for identifying a member of the FRILfamily of progenitor cell preservation factors, comprising contacting acandidate compound with a glycosylated extracellular domain of an FLT3receptor, wherein the glycosylation pattern of the extracellular domainof the FLT3 receptor is the same as the glycosylation pattern of anextracellular domain of a normally glycosylated FLT3 receptor, wherein acandidate compound that binds the glycosylated extracellular domain ofthe FLT3 receptor is identified as a FRIL family member.
 85. The methodof claim 84, wherein the candidate compound is a lectin.
 86. The methodof claim 85, wherein the lectin is synthetic.
 87. The method of claim85, wherein the lectin is from a legume.
 88. A method for isolating apopulation of progenitor cells, comprising contacting a population ofcells with a plurality of FRIL family member molecules, and separatingthe unbound cells, wherein the cells bound to the FRIL family membermolecules are an isolated population of progenitor cells, wherein theFRIL family member molecules comprise a protein that: (1) binds to anormally glycosylated FLT3 receptor; (2) has at least 95% amino acidsequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO:8; and (3)preserves hematopoietic progenitor cells.
 89. The method of claim 88,wherein the isolated population of progenitor cells is from a human. 90.The method of claim 88, wherein the FRIL family member molecules aredetectably labeled.
 91. The method of claim 88, wherein the FRIL familymember molecules are immobilized on a solid support.
 92. The method ofclaim 91, wherein the solid support is a bead.
 93. The method of claim92, wherein the bead is magnetic.
 94. The method of claim 88, whereinthe unbound cells are separated by applying a magnet to the populationof cells contacted with the FRIL family member molecules immobilized onthe magnetic bead.
 95. The method of claim 93, wherein the population ofcells bound to the FRIL family member molecules immobilized on amagnetic bead are rinsed with a physiologically acceptable solutionwhile the magnet is applied.
 96. The method of claim 91, wherein thesolid support is the bottom of a tissue culture plate.
 97. The method ofclaim 88, wherein the isolated population of progenitor cells is apopulation of hematopoietic progenitor cells.
 98. The method of claim88, wherein the population of cells is selected from the groupconsisting of whole blood, umbilical cord blood, bone marrow cells, andfetal liver cells.
 99. The method of claim 88, wherein the population ofcells is a sorted population of cells, wherein a cell of the sortedpopulation does not express a cell surface molecule selected from thegroup consisting of CD11b, CD11c, and CD38.
 100. The method of claim 99,wherein the sorted population of cells is sorted by flow cytometry or bymagnetic bead selection.
 101. An isolated population of progenitor cellsisolated by the method of claim
 88. 102. The isolated population ofprogenitor cells of claim 101, wherein the isolated population ofprogenitor cells is from a human.
 103. The cells of claim 101, whereinthe cells of the isolated population of progenitor cells do not expressCD34.
 104. The cells of claim 101, wherein the cells of the isolatedpopulation of progenitor cells express a receptor tyrosine kinaseselected from the group consisting of from FLK1, FLT1, FLT3, FLT4, andKit.
 105. The cells of claim 101, wherein the cells of the isolatedpopulation of progenitor cells express a cell surface molecule selectedfrom the group consisting of CD11b and CD11c.
 106. The isolatedpopulation of progenitor cells of claim 101, wherein the cells of theisolated population of progenitor cells express FLT3.
 107. The isolatedpopulation of progenitor cells of claim 101, wherein the cells of theisolated population of progenitor cells are selected from the groupconsisting of hemangioblasts, a messenchymal stem cells, bone progenitorcells, hepatic progenitor cells, endothelial progenitor cells,hematopoietic progenitor cells, embryonal stem cells, brain progenitorcell, and dendritic progenitor cells.
 108. The isolated population ofprogenitor cells of claim 101, wherein the cells of the isolatedpopulation of progenitor cells are hematopoietic progenitor cells. 109.The isolated population of progenitor cells of claim 101, whereintransplantation of isolated population of progenitor cells into ananimal lacking a population of hematopoietic progenitor cells sufficientto enable survival of the animal reconstitutes the animal, wherein thetransplanted animal survives.